Study On Allogeneic Bone Marrow Transplantation Immune Tolerance Induced By Recipient-derived Transforming Growth Factor-β1 Treated Dendritic Cells

Allogeneic bone marrow transplantation (allo-BMT) is a potential curative approach to treat malignant and hereditary hematological as well as immunological diseases. To date, acute graft-versus-host disease (GVHD) remains the most severe complication that greatly limits the application and efficacy of allo-BMT. Despite progress in understanding the mediators involved in acute GVHD, treatment has remained very frustrating.Acute GVHD fundamentally depends on donor T cells interaction with antigen presenting cells (APCs) and their subsequent activation, proliferation, and differentiation. This process occurs during the second step of the afferent phase of acute GVHD. Although alloantigen can be presented directly by host-derived and indirectly by donor-derived APCs, host-derived APCs appear to be critical in inducing acute GVHD.Dendritic cells (DCs) are the most potent APCs and play a critical role in the initiation and regulation of immune responses, and are instrumental in the induction as well as maintenance of tolerance. The ability of DCs to induce immunity or tolerance appears to be related to their state of functional maturation. In contrast to mature DCs (mDCs) that stimulate T cells through high expression of MHC II and costimulatory molecules, immature DCs (imDCs) inhibit T cell responses and induce tolerance. The immune regulatory aspects of imDCs include the directkilling of T cells, induction of T cell anergy or stimulation of T regulatory cell (Treg) generation.Transforming growth factor β1 (TGF-β1) is a secreted protein that regulates proliferation, differentiation and death of various cell types. Previous reports had showed that TGF-β1 could inhibit DCs maturation, and TGF-β1-treated DCs (TGFβ-DCs) could prolong the cardiac allograft suvival in fully MHC-mismatched murine heart transplantation.In the current study, recipient-derived TGFβ-DCs were adoptive transfused mixed with allogeneic hematopoietic stem cells in a murine fully MHC-mismatched allo-BMT model. We hope they can induce specific immune tolerance, which may prevent or relieve acute GVHD and to provide a theoretical and experimental basis for designing new preventive and therapeutic strategies for acute GVHD.There were three sections in this study.Section 1: Study on effects of transforming growth factor-pi on dendritic cellsC57BL/6J murine bone marrow cells were cultured with different cytokines combination to generate immature DCs (imDCs, GM-CSF only) and TGFβ-DCs (GM-CSF±TGF-β1). Afterwards, they were stimulated by lipopolysaccharide (LPS). After culturing for 5 days, TGFβ-DCs and imDCs exhibit an immature characteristic DCs morphology. They appeared mainly as needle-like processes and prominent endocytic compartment. LPS stimulation promoted the development of typical cellular protrusions on imDCs, but TGFβ-DCs had no significant alterations in ultrastructure after LPS stimulating and maintained immature properties. The phenotypes of DCs were analyzed by flow cytometry (FCM). Both TGFβ-DCs and imDCs expressed mouse BM-derived DCs specific marker CD11c. Surface expression of CD80, CD86, CD40, I-Ab were inhibited by addition of TGF-β1, especially in CD80, CD86 (4.14±0.95% vs 13.90 ± 7.22%; 8.60±0.75% vs 20.63 ± 5.03%, P<0.05, both). Furthermore, the imDCs were sensitive to further maturation in response to LPS by showing increased levels of MHC class II, CD80, CD86 and CD40. In marked contrast, TGFβ-DCs were resistant to maturation by LPS, as the expressions of I-Ab, CD80 was slightly increased(41.33±4.67% vs 12.17 ± 1.64%, p<0.01 and 33.47 ± 6.77% vs 6.53 ± 5.43%, P<0.05).After 96 h mixed lymphocyte reaction (MLR), TGFβ-DCs had weaker stimulating capacity than imDCs, especially when the ratio of DCs and T cells were 1:4 and 1:1(△A value: 0.286 ±0.037 vs 0.554±0.059, p<0.05 and 0.323±0.031 vs 1.035±0.113, P<0.01). On the contrary, significantly allostimulatory activity was seen in mDCs. Importantly, maturation induced by LPS stimulation strongly promoted the allostimulatory capacity of imDCs, whereas exposure to LPS only slightly affected that of TGFβ-DCs. This observation indicated that TGFβ-DCs were partially maturation resistant. BALB/c allogenic T cells were co-cultured with C57BL/6J derived TGFβ-DCss for 3 days, and then restimulated for a further 4 days with C57BL/6J spleen cells or standard dose ConA. T cells cultured with TGFβ-DCs or imDCs both were unresponsive to the titrated amounts of spleen cells, but imDCs pretreated T cells were stronger than TGFβ-DCs, especially when stimulating cells/responding cells were 1:10 and 1:1(△A value: 0.429 ± 0.079 vs 0.261±0.037, p<0.05 and 0.635±0.058 vs 0.346 ± 0.048, p<0.01). However, both two T cell populations proliferated in response to ConA as a unspecific polyclonal stimulus, suggesting that the TGFβ-DCs had induced a state of alloantigen-specific T cell unresponsiveness.After treatment of imDCs and TGFβ-DCs with LPS for 24 h, the production of IL-12p70 of TGFβ-DCs was significantly less than that of imDCs(115.4±15.2 pg/ml vs 517.0±29.7 pg/ml, p<0.01), but the level of Th2 cytokine-IL-10 was elevated(132.1 ± 17.5 pg/ml vs 75.1 ± 16.6 pg/ml, P<0.05), indicating that exposure to TGF-β1 impaired the capability of DCs to produce high amounts of bioactive IL-12p70. According to the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the expressions of chemokine MIP-1a mRNA on TGFβ-DCs after LPS stimulation were lower than imDCs at each time point. The lower expressions of MCP-1 and IP-10 on TGFβ-DCs at irregular pattern after LPS treatment, whereas the expressions RANTES were no different.Our results showed strong inhibitory effect of TGF-β1 on DCs maturation, and TGFβ-DCs were resistance to maturation stimulus (LPS) in culture.Section 2: Transforming growth factor-pi inhibits the maturation of dendritic cells via Toll-like receptor 4 signaling pathwayIt had been previously demonstrated that LPS is recognized by Toll-like receptor 4(TLR4) and LPS can stimulate imDCs to mDCs via TLR4 signaling pathway. So we explored the effects of TGFβ1 on expression of TLR4 on DCs.We analyzed the expression of TLR4 mRNA on imDCs and TGFβ-DCs by RT-PCR. To our surprise, although DCs clearly expressed TLR4, TGFβ-DCs expressed more weakly than imDCs(TLR4/β-actin ratio: 1.16±0.183 vs 0.419±0.087, P<0.05). The expressions of TLR4 on DCs were also estimated by FCM. Consistent with the result of RT-PCR, the positive expression percentage and mean fluorescence intensity of TLR4 on imDCs were higher than those on TGFβ-DCs(51.8 ± 3.89% vs 15.7±4.13%, P<0.01 and 2.37±0.26 vs 1.36±0.17, p<0.05). The results agreed with previous findings that TGFβ-DCs responded weakly to LPS. We further estimated the activities of nuclear factor-κB (NF-κB), ERK1/2 and p38 proteins involved in TLR4 signaling pathway. Using EMSA, we found the NF-kB DNA binding activity in imDCs was significantly increased in response to LPS, but addition of TGF-β1 to DCs inhibited NF-kB binding(860±154 vs 3555±1124, P<0.01). Moreover, TGF-β1 was effective in suppressing LPS-induced activation of ERK1/2 and p38 kinase, the level of phosphorylation of ERK1/2 and p38 kinase were lower than imDCs measured by Western Blot (0.318 ± 0.316 vs 0.909±0.109, P<0.01 and 0.471±0.071 vs 0.975± 0.118, P<0.05).The results suggested TGF-β1 maybe directly inhibit TLR4 expression on DCs, and then interfere with the activity of downstream key proteins, such as NF-kB, ERKl/2 and p38. Ultimately, TGF-β1 treated DCs were resistance to LPS, down-regulated the expression of costimulatory molecular on DCs, decrease the secretion of inflammatory cytokines.Section 3: Induction of specific immune tolerance in murine allo-BMT model by recipient-derived transforming growth factor-β1 treated dendritic cellsEvidences had accumulated the host APCs play a central role in the initiation of acute GVHD. Others and we have found TGFβ-DCs induced allogeneic specific immune tolerance in vitro. So in the current study, we focus on the effect of recipient-derived TGFβ-DCs on murine fully MHC-mismatched allo-BMT model.5.0×106 recipient-derived TGFβ-DCs were injected into female C57BL/6 (H-2b) with bone marrow-splenocyte (BMS) grafts from MHC disparate male donor BALB/c mice (H-2d). Survival and pathology analysis showed mean survival time (MST) of untreated BMT recipient was 9.5±0.6 days, while TGFP-DCs co-transplantation resulted in significant prolongation of survival and MST was 44.3 ± 4.5 days (P<0.01). However, mature DCs aggravated the acute GVHD and the MST was only 6.6±0.6 days (P<0.01). In addition, the donor-derived and third party-C3H (H-2k)-derived TGF+-DCs as well as recipient-derived imDCs could not enhance the survival time(11.3±0.6 days, 9.7±0.5 days, and 11.2±0.7 days; P>0.05, all). To better understand the half-life of the infected TGFβ-DCs,the recipients derived TGFβ-DCs were labeled with CFSE-DA, a fluorescence trace detector. We detected approximately 5% of CFSE-Iabeled TGFβ-DCs in spleen mononuclear cells the next day after transplantation and the half-life of the injected TGFβ-DCs was around 16 days after transplantation.Furthermore, serum IFN-γ, IL-12 and IL-18 levels in TGFβ-DCs co-transplantation mouse reduced compared with untreated BMT mouse (173.8 ±34.3 pg/ml vs 296.0±32.3 pg/ml; 102.8±18.6 pg/ml vs 290.3±59.0 pg/ml; 88.0±37.2 pg/ml vs 479.0±39.8 pg/ml; p<0.05, all), while serum IL-10 level was not changed (208.5 ± 55.9 pg/ml vs 185.3 ± 31.3 pg/ml, P>0.05). CD4+CD25+ regulatory T (Treg) cells are supposed to maintain immunologic self-tolerance. The percentages of Treg cells were increased in splenocyte CD4+ T cells started from 5 days after transplantation, indicating TGFβ-DCs induce the generation of Treg cells in host (15.61 ±2.08% vs 11.67±1.16%, p<0.05). Notably, about 85% Y chromosome from male donor were found in recipients bone marrow cells 30 days after transplantation, and about only 3.2% host H-2b type cells in peripheral blood cells, indicating that TGFβ-DCs did not prevent donor cell engraftment under these experimental conditions, and the attenuation of acute GVHD in recipients derived TGFβ-DCs was not attributed to poor engraftment of donor cells with concomitant autologous recovery. In delayed type hypersensitivity (DTH) assay, the long-time survival recipient mice (±60 d) sensitized with allogeneic donor splenocyte showed lower reactivity than that in none-BMT group(0.30±0.04mm vs 0.56±0.03mm, P<0.0l), but still presented higher reactivity to third party C3H mice (0.62±0.07mm vs 0.56±0.03mm, p>0.05), suggesting the tolerance were durable and specific.Summary: (1) TGF-β 1 inhibited DCs maturation and TGFβ-DCs could induce specific allogeneic T cells hyporesponsiveness in vitro (2) Compared with imDC, TGFβ-DCs were resistant to maturation with LPS. Maturation resistance was evident from a failure to up-regulate co-stimulatory molecules, to stimulate stronger T cells proliferation, to enhance secretion of IL-12p70 and expressions of MIP-1a, MCP-1 and IP-10 mRNA. (3) TGF-β 1 maybe directly inhibit LPS recepotor-TLR4 expression on DCs, and then suppress the activity of downstream key proteins, such as NF-kB, ERKl/2 and p38. (4) Adoptive transfusion of recipient-derived TGFβ-DCs could attenuate acute GVHD, and induce MHC-specific tolerance in mice, but donor, third party derived TGFβ-DCs have no significant effects on recipients mice survival. (5) The protection effects of recipient-derived TGFβ-DCs maybe mediated by suppressing Th1 responses and inducing Treg cells.