Liver allograft rejection in rats depleted of CD8+ cells

The mechanism(s) of rejection or tolerance induction is a competitive, complex process that presumably involves interactions between multiple subpopulations of T lymphocytes. We investigated the roles of CD8⁺ cytolytic and CD4+ helper T cells in rat strains that tolerate liver allografts and that differ at both the major histocompatibility complex (MHC) (RT1) and minor histocompatibility genes. Orthotopic liver transplantation (OLT) with arterial reconstruction was performed with Brown Norway (BN) (RT1ⁿ) donors and Lewis (RT1¹) recipients, some of which were untreated, others treated with anti-CD8 antibody, and still others treated with anti-CD4 antibody. Liver graft rejection was monitored for 28 days on the basis of two criteria: (1) serum levels of AST enzyme at 3-day intervals and (2) liver biopsies at weekly intervals and at the time of sacrifice at the end of the study period. In the untreated control group, an elevation of AST was found to peak at day 6 after grafting, and it remained elevated until day 28 (AST 542±72 U/l). Histologically, signs of severe rejection were first observed on day 9; these changed to moderate rejection about day 21 and to mild rejection by day 28, when the animals were sacrificed. Recipients pre-treated with anti-CD8 demonstrated a significant elevation of AST within 6 days that, unlike in the control recipients, continued to rise sharply through the observation period (AST 1127±181 U/1, P=0.009 vs control group). Liver biopsies showed mild rejection at day 9 and moderate rejection at days 21 through 28. Recipients pretreated with anti-CD4 showed a time course of enzyme elevation and severity of rejection that was not significantly different from that observed in the control group. However, anti-CD4 treatment resulted in only 75% depletion of CD4⁺ cells in peripheral blood as compared to complete elimination of CD8⁺ cells following anti-CD8 treatment. Functional studies of spleen and liver-infiltrating lymphocytes obtained after 28 days showed low proliferative response in mixed lymphocyte culture with both BN and PVG stimulator spleen and lymph node cells. These results suggest that in this donor/recipient combination, removal of CD8⁺ cells increases the severity of rejection as demonstrated by a progressive rise in AST and histology. Moreover, OLT in this combination results in a profound, nonspecific inhibition of proliferative T-cell responses to MHC alloantigens.     

Induction of transplantation tolerance—the potential of regulatory T cells

Solid organ transplantation is widely accepted as an effective treatment for end organ failure. Although the treatment with immunosuppressive drugs has undoubtedly greatly improved graft survival, chronic rejection still has considerable impact on long term outcome. This, together with the undesirable side effects associated with life long treatment with immunosuppressive drugs, have significant implications for long term outcomes.In a small number of patients, drug non-compliance as well as controlled reduction or removal of maintenance immune suppressive drug therapy has led to the uncovering of a tolerant state. The challenge of achieving improved monitoring of all transplant patients may allow tailoring of immunosupression in a proportion of recipients thereby increasing the opportunities for the induction of specific unresponsiveness to donor alloantigens in the future. The immune system using several mechanisms to both induce and maintain tolerance to alloantigens, including the deletion of allo-reactive T cells, the induction of anergy, clonal exhaustion, ignorance and active suppression (immunoregulation) of allo-responses. A minor subpopulation of CD4⁺ T cells, regulatory or suppressor CD4⁺ T cells that co-express the cell-surface molecule CD25 (IL2 α subunit) at a high level may play a major role in the maintenance of specific unresponsiveness and operational tolerance to donor antigens in vivo. Intensive investigation of these cells in recent years has started to uncover the mechanisms of active suppression by regulatory T cells in this setting.     

Prognostic value of peripheral blood T lymphocyte subsets in clear cell renal cell carcinoma

BackgroundTo date, few studies have evaluated the role of peripheral blood T lymphocyte subsets in patients with clear cell renal cell carcinoma (ccRCC). Here we measured the levels of peripheral blood T lymphocyte subsets and evaluated its prognostic value in ccRCC.MethodsData from 122 patients with RCC from January 2018 to January 2020 were collected. Preoperative peripheral blood T lymphocyte subsets and medical records were analyzed. Kaplan-Meier cures and log rank test were used for analyzing overall survival (OS). Univariate and multivariate survival analyses were underwent by performing the Cox proportional hazards models. Correlations were tested by Pearson’s correlation analysis.ResultsOf 122 patients, a total of 80 ccRCC patients was enrolled. Patients with low CD3⁺ T cells and low CD4⁺/CD8⁺ ratio displayed a worse OS than patients with high CD3⁺ T cells and high CD4⁺/CD8⁺ ratio (P=0.029 and 0.002, respectively). Multivariate analyses showed CD3⁺ T cells and CD4⁺/CD8⁺ ratio were independent predictive factors for the OS (HR: 0.295, 95% CI, 0.091–0.956; P=0.042 and HR: 0.244, 95% CI, 0.065–0.920; P=0.037, respectively). Moreover, NLR negatively correlated with both levels of CD3⁺ T cells and CD4⁺/CD8⁺ ratio (P<0.001, r=−0.398 and P=0.012, r=−0.280, respectively).ConclusionsThe findings of our study suggest that preoperative CD3⁺ T cells and CD4⁺/CD8⁺ ratio in peripheral blood are independent predictors for patients with ccRCC.     

Th17/Treg cell balance in stable liver transplant recipients

BackgroundImmune monitoring of transplanted patients may provide a reliable basis for the individualization of immunosuppressive therapy. In addition, it might be applied for realizing the early and non-invasive diagnosis of acute allograft rejection.MethodsPercentages of TCD4 + IL-17+ (Th17) and TCD4 + CD25 + CD127^dim/− (Treg) cells, as well as serum levels of interleukin (IL)-17 and transforming growth factor (TGF)-β1, were evaluated in 30 stable patients using flow cytometry and ELISA techniques before and six months after liver transplantation. Besides, the same cells and cytokines were quantified in 10 recipients with acute allograft rejection.ResultsSix months post-transplant, the percentage of Th17 and Treg cells in the peripheral blood of stable liver transplant recipients reduced significantly, but the Th17/Treg ratios were comparable to the pre-transplant period (1.24 vs. 1.56); however, Th17/Treg ratios in the rejection group was significantly higher than in the stable recipients (4.06 vs. 1.56, P-value = 0.001). Stable patients showed decreased amounts of serum IL-17 which was remarkably lower than in the rejection group (P-value = 0.01). Moreover, there was a significant correlation between the serum level of IL-17 and the percentage of Th17 cells (P-value <0.001). Th17 frequency was negatively associated with the liver allograft function. Notably, TGF-β1 levels differed neither between pre-and post-transplant samplings nor between stable and rejection groups.ConclusionSix months after liver transplantation, the mean Th17/Treg ratio in stable recipients remained comparable to the pre-transplant values; however, it was significantly elevated in patients with acute allograft rejection, suggesting the Th17/Treg ratio as a probable predictor of acute rejection.     

Gut microbiota intervention by pre and probiotics can induce regulatory T cells and reduce the risk of severe acute GVHD following allogeneic hematopoietic stem cell transplantation

BackgroundAcute graft-versus-host disease (aGVHD) is one of the leading causes of limitation and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Numerous studies have shown that changes in the gut microbiome diversity increased post-transplant problems, including the occurrence of aGVHD. Probiotics and prebiotics can reconstitute the gut microbiota and thus increase bacterial metabolites such as short-chain fatty acids (SCFAs) that have immunomodulatory effects preventing aGVHD in recipients of allo-HSCTs.Methods/Study DesignWe conducted a pilot randomized clinical trial to investigate whether oral synbiotics are associated with the prevention or reduction in occurrence/severity and mitigate complications of aGVHD following allo-HSCT. A commercially available synbiotic mixture containing high levels of 7 safe bacterial strains plus fructo-oligosaccharides as a prebiotic was administered to allo-HSCT recipients. Out of 40 allo-HSCT patients, 20 received daily a synbiotic 21 days prior to transplantation (days −21 to day 0). In contrast, in the control group 20 recipients of allo-HSCT did not receive a symbiotic therapy.ResultsWithin first 100 days of observation, the incidence of severe (grade III/IV) aGVHD in the a synbiotic-therapy group was 0% (0 out of 20 patients), whereas it was 25% (5 out of 20 patients) in the control group (P = 0.047). The median percentage of CD4 + CD25 + Foxp3+ regulatory T cells (Tregs) among CD4+ lymphocytes on day 28 after HSCT in the synbiotic group was higher (2.54%) than in control group (1.73%; P = 0.01). There was no difference in Treg cells on day 7 after HSCT between two groups. However, the median percentage and the absolute count of Tregs in patients who experience aGVHD was significantly lower on days 7 and 28 after HSCT (both P < 0.05). The overall 12-month survival (OS) rate was higher (90%) in the symbiotic-treated patients than in the control group (75%), but the difference was not statistically significant (P = 0.234).ConclusionOur preliminary findings suggest that synbiotic intake before and during the conditioning regimen of allo-HSCT patients may lead to a reduction in the incidence and severity of aGVHD through the induction of CD4 + CD25 + Foxp3+ regulatory T cells, thus contributing to the improvement of transplant outcomes. Much larger studies are needed to confirm our observations.     

Double-Negative αβ T Cells Are Early Responders to AKI and Are Found in Human Kidney

Ischemia-reperfusion injury (IRI) is a major cause of AKI, and previous studies established important roles for conventional CD4⁺ T cells, natural killer T cells, and CD4⁺CD25⁺FoxP3⁺ Tregs in AKI pathogenesis. We recently identified CD4⁻CD8⁻ (double-negative; DN) T cells as an important subset of αβ T cell receptor–positive cells residing in mouse kidney. However, little is known about the pathophysiologic functions of kidney DN T cells. In this study, we phenotypically and functionally characterized murine kidney DN T cells in the steady state and in response to IRI. Unlike CD4⁺ and CD8⁺ T cells, DN T cells in the steady state expressed high levels of CD69, CD28, and CD40L; differentially expressed IL-27 and IL-10 anti-inflammatory cytokines; spontaneously proliferated at a very high rate; and suppressed in vitro proliferation of activated CD4⁺ T cells. Within the first 3–24 hours after IRI, kidney DN T cells expanded significantly and upregulated expression of IL-10. In adoptive transfer experiments, DN T cells significantly protected recipients from AKI by an IL-10–dependent mechanism. DN T cells also made up a large fraction of the T cell compartment in human kidneys. Our results indicate that DN T cells are an important subset of the resident αβ⁺ T cell population in the mammalian kidney and are early responders to AKI that have anti-inflammatory properties.     

异基因造血干细胞移植受者外周血中可溶性HLA-G与急性GVHD的相关性

目的 探讨异基因造血干细胞移植(allo-HSCT)受者外周血中可溶性HLA-G(sHLA-G)的水平与急性移植物抗宿主病(aGVHD)间的相关性以及与CD4+CD25~(hlgh)调节性T淋巴细胞(Treg)水平的相关性.方法 选择27例allo-HSCT受者,将其中13例术后发生aGVHD者作为aGVHD组,14例未发生aGVHD者作为对照组.在移植预处理前、移植后第2、4、8、14周时,分别抽取两组受者清晨空腹肘静脉血2～4 ml,采用酶联免疫吸附试验双抗夹心法定量检测sHLA-G,采用流式细胞术检测CD4+CD25+Treg.动态监测术后第2和14周受者sHLA-G水平的变化;动态监测和分析术后第8和14周两组受者间sHLA-G的差异.观察sHLA-G与发生aGVHD的相关性;检测术后第14周两组受者CD4+CD25+Treg的数量,观察sHLA-G水平与CD4+CD25+Treg的相关性.结果 预处理前,对照组和aGVHD组受者sHLA-G的水平分别为(75.4±13.1)/μg/L和(47.0±14.1)btg/L,两组间差异有统计学意义(P<0.05).术后第8周和第14周,对照组受者sHLA-G的水平明显高于相应时间点aGVHD组受者,两组间差异均有统计学意义(P<0.01).对照组受者术后第14周的sHLA-G水平与第2周相比,增长了1.2～3.4倍,两时间点的差异有统计学意义(P<0.05).术后第14周,通过Spearman等级相关检验显示受者sHLA_G水平与CD4+CD25+Treg比例具有统计学上的相关性(相关系数r=0.810,P<0.05),结论 allo-HSCT后受者体内sHIA-G对aGVHD的发生起负性调节作用;sHLA-G的水平与外周血CD4+CD25~(hlgh) T淋巴细胞的比例旱正相关,二者之间可能存在相互诱导和调节的信号途径,在诱导和维持移植免疫耐受中起着重要的协同作用.     

Induction of transplantation tolerance by allogeneic donor-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells

Several studies have shown that recipient-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are involved in transplantation tolerance. However, it is not clear whether allogeneic donor-derived Tregs are able to regulate T cell alloreactivity after solid organ allograft transplantation. Related studies in experimental bone marrow transplantation have shown that allogeneic donor-derived Tregs are capable of promoting early and long-term allogeneic hematopoietic engraftment, accompanied by tolerance to donor and recipient antigens. However, in these models, donor-derived Tregs are syngeneic with respect to the T responder cells. The role of Tregs in solid organ transplantation models where recipient-derived T responder and donor-derived Tregs are allogeneic has been scarcely studied. In order to determine whether allogeneic Tregs were able to regulate T cell alloreactivity, CD4(+)CD25(-) and CD8(+) T responder cells were cultured with stimulator dendritic cells in several responder-stimulator strain combinations (C57BL/6-->BALB/c, BALB/c-->C57BL/6 and C3H-->BALB/c) in the presence of responder-derived, stimulator-derived or 3rd-party-derived Tregs. Then, the frequency of IFN-gamma+ alloreactive T cells was determined by means of ELISPOT assay. The results of this study demonstrate that, regardless of the responder-stimulator strain combination, both responder-derived and stimulator-derived Tregs, but not 3rd-party-derived Tregs, significantly inhibited CD4(+) and CD8(+) T cell alloreactivity. The effect of allogeneic stimulator-derived Tregs was dependent on IL-10 and TGF-beta and reversed by exogenous IL-2. In vivo experiments in nu/nu recipients reconstituted with CD4(+)CD25(-) T responder and Tregs showed that recipient and donor-derived, but not 3rd-party-derived Tregs, significantly enhanced skin allograft survival. Importantly, T cells from both recipient-derived and donor-derived Treg-reconstituted nu/nu recipients exhibited donor-specific unresponsiveness in vitro. These results show that allogeneic donor-derived Tregs significantly inhibit T cell alloreactivity and suggest their potential use in the induction of transplantation tolerance.     

Apoptosis of Peripheral CD4+ T-Lymphocytes in End-Stage Renal Disease Patients Under Hemodialysis and rhEPO Therapies

End-stage renal disease (ESRD) under hemodialyses (HD) is related with a higher propensity to infections, essentially due to T-cell lymphopenia. We postulated that HD procedure affects CD4⁺ T cells, especially by inducing apoptotic death and that recombinant human erythropoietin (rhEPO) therapy may also play an important role in the modulation of the immune system in these patients. T-cell phenotype and apoptosis of HD patients and healthy controls were evaluated by flow cytometry using anticoagulated whole-blood samples. In 12 HD patients, these parameters were also analyzed before and immediately after HD procedure. HD patients showed a decrease in total circulating CD3⁺ lymphocytes, especially in CD4⁺ T cells (0.747 ± 0.410 vs. 0.941 ± 0.216 × 10⁹/L, p < 0.05), which could be a consequence of the higher proportion of CD3⁺ and CD4⁺ lymphocytes in the latest stage of apoptosis (or death) and of the higher proportion of apoptotic CD4⁺ T cells observed in the patients immediately after HD procedure (2.91 ± 0.780 vs. 3.90 ± 1.96, p < 0.05). A positive and statistically significant correlation between CD3⁺ and CD4⁺ lymphocytes in latest stage of apoptosis (or death) with HD time was found (CD3⁺: r = 0.592, p < 0.01; CD4⁺: r = 0.501, p < 0.01). We also found a negative and significant correlation between weekly rhEPO doses and the number of CD4⁺ T cells (r = –0.358, p < 0.05). In conclusion, HD procedure still contributes to the development of T-cell lymphopenia, at least in part, by apoptosis induction. It was also shown that rhEPO therapy is associated with the CD4⁺ T-cell decline, possibly by immune modulation, eliminating atypical cells and helping to restore the CD4⁺ T-cell subset.     

Immune-mediated liver injury: prognostic value of CD4+, CD8+, and CD68+ in infants with extrahepatic biliary atresia

Background

Hepatic fibrosis and cirrhosis develop progressively in extrahepatic biliary atresia (EHBA) despite timely surgical intervention.

Purpose

The aim of the study was to define CD4⁺ helper T lymphocytes, cytotoxic CD8⁺ T lymphocytes, and CD68⁺ (macrophages) infiltration of portal tracts and lobules and hepatic fibrosis as possible predictive measures of outcome of infants having EHBA.

Methods

The outcome of 32 infants with EHBA was correlated to their percutaneous biopsy and postportoenterostomy core liver tissue infiltration by CD4⁺, CD68⁺, and CD8⁺ cells and to the degree of detected fibrosis.

Results

Portoenterostomy cores were heavily infiltrated by CD4⁺, CD8⁺, and CD68⁺, compared with the preoperative liver biopsy (P = .008, .004, and .017, respectively). Infants having favorable outcome had more macrophage infiltration in portoenterostomy core compared with those having an unfavorable outcome (25.66 ± 29.77 per HPF compared with 11.62 ± 4.58, P = .000). Mean CD4⁺/CD8⁺ ratio was 1.54 ± 1.37 in those who died within 18 months postoperatively and 0.733 ± 0.48 in others (P = .021).

Conclusion

Immune-mediated destruction of portal tracts is an integral part of pathogenesis of EHBA.     

Correlation of the CD4+CD25high T-regulatory cells in recipients and their corresponding donors to acute GVHD

Graft-versus-host disease continues to be a major life-threatening complication after allogeneic hematopoietic stem cells transplantation (aHSCT). The relationship of acute GVHD (aGVHD) with the levels of peripheral CD4(+)CD25(high) T cells in patients after aHSCT and in their corresponding donors is not fully investigated. We examined the levels of CD4(+)CD25(high) T cells in patients after aHSCT and in their corresponding donors, and analyzed the relationship of CD4(+)CD25(high) T cells to the incidence and prognosis of aGVHD. The recipients with normal or high CD4(+)CD25(high) T cells (three of eight, 37.5%) had no or mild aGVHD (grade I), and all survived during the follow-up period. In striking contrast, the recipients with lower or no CD4(+)CD25(high) T cells suffered from greater than grade II aGVHD (four of four, 100%), and all died within 1-year post-aHSCT. Moreover, the number of CD4(+)CD25(high) T cells in recipients correlated significantly with that of their corresponding donors. The CD4(+)CD25(high) T cells from the recipients and their corresponding donors expressed high levels of Foxp3, and effectively suppressed the proliferation of CD4(+)CD25(-) responder T cells. This study suggests that human Treg cells may play an important role in aGVHD, as has been seen in murine models. The levels of peripheral CD4(+)CD25(high) T cells in recipients and donors could be helpful for predicting of the onset and outcome of aGVHD.     

Augmented bacterial elimination by Kupffer cells after IL-18 pretreatment via IFN-γ produced from NK cells in burn-injured mice

We recently demonstrated that IL-18 injections following burn restored IFN-γ production and increased mouse survival after bacterial infection. However, it has yet to be fully elucidated how the IL-18 therapy affects the function of phagocytic cells. We investigated the effect of IL-18 therapy on function and interaction of Kupffer cells and NK cells in burned mice. C57BL/6 mice received a 20% full-thickness burn, followed by multiple injections with IL-18. Although burn-injured mice had decreased expression of IL-18 receptors on the NK/NKT cells 5 days after injury, multiple IL-18 injections restored this expression. IL-18 treatment also augmented Kupffer cell phagocytosis. Although burn decreased the number of CD68⁺ Kupffer cells with phagocytic activity, IL-18 treatment partially restored their proportion, and augmented phagocytosis-induced ROS production in CD68⁺ Kupffer cells after the injection of heat-killed Escherichia coli. Consistently, IL-18 restored the impaired E. coli killing activity of Kupffer cells of burn-injured mice. Such Kupffer cell activation by IL-18 was abrogated by the deletion of NK cells or IFN-γ. In conclusion, IL-18 therapy in burn-injured mice enhanced function of CD68⁺ Kupffer cells via the activation of liver NK cells and augmentation of their IFN-γ production, thereby improving survival after E. coli infection.     

Suppressive effects of fisetin on mice T lymphocytes in vitro and in vivo

Background

Most of the immunosuppressive drugs have satisfactory therapeutic effects on organ transplantation and autoimmune disease. However, their clinical application is limited by side effects. Therefore, new and safe immunosuppressive drugs against acute and chronic rejections are eagerly awaited. Fisetin, a flavonoid present in various types of vegetables and fruits, has few side effects and low level of toxicity, which would be a desirable clinical feature. In the present study, we investigated the immunosuppressive effects and underlying mechanisms of fisetin against T-cell activation in vitro and in vivo.

Methods

We measured the effect of fisetin on T-lymphocyte proliferation, T-cell subsets, cell cycle progression, cytokine production, and nuclear factor activation in vitro, as well as its influence on T cell–mediated delayed-type hypersensitivity reaction in vivo.

Results

In vitro, the results showed that fisetin significantly suppressed mouse splenocytes proliferation, Th1 and Th2 cytokine production, cell cycle and the ratio of CD4⁺/CD8⁺ T cells. Furthermore, fisetin exerts an immunosuppressive effect in mouse T lymphocytes through the suppression of nuclear factor kappa B activation and nuclear factor of activated T cells signaling in a dose-dependent manner. In vivo, fisetin treatment also significantly inhibited the dinitrofluorobenzene-induced delayed-type hypersensitivity reactions in mice.

Conclusions

Fisetin had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for fisetin as an immunosuppressive agent.     

Soluble FGL2 induced by tumor necrosis factor-α and interferon-γ in CD4 T cells through MAPK pathway in human renal allograft acute rejection

Background

Acute rejection (AR), initiated by alloreactive CD4⁺ T cells, hampers allograft survival. Soluble fibrinogen-like protein 2 (sFGL2) is a novel effector of CD4⁺ T cells. We previously found that serum sFGL2 significantly increased in renal allograft recipients with AR. In this study, sFGL2 secretion by CD4⁺ T cells and its mechanism were further explored both in vivo and in vitro.

Materials and methods

Forty cases of living-related renal transplant recipients with biopsy-proven AR or stable renal function were collected and detected serum sFGL2, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and peripheral CD4⁺ T cells. In vitro, the isolated human CD4⁺ T cells were stimulated by TNF-α or IFN-γ. sFGL2 in the supernatant and mitogen-activated protein kinase (MAPK) proteins in the CD4⁺ T cells were investigated. Approval for this study was obtained from the Ethics Committee of Fudan University.

Results

sFGL2, TNF-α, IFN-γ, and CD4⁺ T cells were significantly increased in the peripheral blood of renal allograft recipients with AR. Stimulation with 1000 U/mL TNF-α or 62.5 U/mL IFN-γ for 48 h provided an optimal condition for CD4⁺ T cells to secrete sFGL2 in vitro. Phosphorylated (p-) c-Jun N-terminal kinase was remarkably upregulated in the activated CD4⁺ T cells, whereas no significant changes were found in p-p38 MAPK or p-ERK1/2 expression. Furthermore, inhibition of c-Jun N-terminal kinase significantly reduced sFGL2 secretion by CD4⁺ T cells.

Conclusions

sFGL2 secretion by CD4⁺ T cells can be induced with TNF-α and IFN-γ stimulation through MAPK signaling in renal allograft AR. Our study suggests that sFGL2 is a potential mediator in the pathogenesis of allograft rejection.     

Studies on the participation of different T cell subsets in rat liver allograft rejection

In this study, we investigated which subsets of rat T cells (CD8 + vs. CD4 + ) are involved in the rejection of liver allografts by the in vivo administration of monoclonal antibody (OX-8 or OX-38, and W3/25 MAb) into thymectomized recipient Lewis (RTI¹) rats prior to DA (RTI^a) liver transplantation. We also compared the results of allograft survival of liver and heart transplants under the same experimental conditions. In order to deplete either CD8 + T cells or CD4 + T cells from recipient animals, 0.4 ml of OX-8 (ascitic form) or a 0.8 ml cocktail of MAb W3/25 and OX-38 (0.4 ml each) was injected into thymectomized recipient rats, respectively. Untreated Lewis rats consistently rejected donor DA liver grafts between 9 and 11 days (n = 7, 9.8 days ± 1.1 days). In contrast, anti-CD8 MAb pretreatment extended the survival times of DA liver grafts for up to 40 days (n = 5, 26.8 days ± 8.4 days). Furthermore, survival of DA liver grafts was significantly prolonged in Lewis rats that had been pretreated with anti-CD4 MAb (n = 7,35.6 days ± 17.9 days). Two out of seven recipient animals survived for more than 60 days. For heart transplantation, untreated Lewis rats rejected DA heart grafts between 6 and 8 days after operation (n = 6, 6.5 days ± 1.2 days). Anti-CD4 MAb treatment prolonged heart graft survival for more than 60 days in all cases (n = 3, > 60 days). However, there was virtually no effect of anti-CD8 MAb treatment on heart graft survival (n = 4, 7.0 days ± 0.9 days). These results suggested that when whole MHC disparity prevailed between donor and recipient, both subsets of T cells were required for the rejection of liver allografts and that class II reactive T cells predominantly mediated liver graft rejection. Furthermore, CD8 + T cells played a differential role in the rejection of rat liver and heart allograft.     

Heat shock preconditioning inhibits CD4+ T lymphocyte activation in transplanted fatty rat livers

Heat shock preconditioning (HPc) of fatty donor livers significantly increases recipient survival in rats. We investigated to what extent the blockade of Kupffer cells by gadolinium chloride (GdCl3) can mimic the effect of HPc and the involvement of liver CD4+ T lymphocytes in HPc. Fatty liver was experimentally induced in Lewis rats by a choline- and methionine-deficient diet. Fatty liver donors were pretreated with HPc (42.5 degrees C for 10 min), the Kupffer cell inhibitor GdCl3, or placebo (sham group). Donors were then harvested, stored in University of Wisconsin preservation solution for 12 h at 4 degrees C, and transplanted into normal syngeneic rats. Hepatic injury (alanine aminotransferase) and serum cytokines (interleukin-12p70, tumor necrosis factor-alpha, and interleukin-10) of recipients increased at 3 h, then decreased, and increased again at 24 h after transplantation. HPc treatment diminished both the early and later phases of this biphasic response and improved recipient survival. GdCl3 reduced these cytokines in the early but not the later phase and did not reduce neutrophil accumulation or improve the recipient survival. HPc, but not GdCl3 treatment, also reduced the number of liver CD4+ T lymphocytes and their interferon-gamma production. We conclude that HPc, but not GdCl3 treatment, prevents biphasic liver injury and the activation of liver CD4+ T lymphocytes in transplanted fatty donor livers.     

Association between CD34+ and CD3+ T-cells in allogeneic grafts and acute graft-versus-host disease in children undergoing allogeneic hematopoietic stem cell transplantation: A single-center study

BackgroundAcute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We examined the association between the composition of the cell subsets present in allogeneic grafts (allografts) and the occurrence and severity of aGVHD in pediatric patients.MethodsWe retrospectively analyzed 80 consecutive pediatric patients undergoing allo-HSCT at our center.ResultsBoth univariate and multivariate analyses showed that the number of CD34⁺ and CD3⁺ T-cells in allografts were the two highest risk factors associated with II–IV aGVHD. Using receiver operating characteristic analysis, the cutoff levels of the allo-HSCT cell doses were used to divide the recipients into low-dose and high-dose groups. The 100-day cumulative incidence of II-IV aGVHD in the high-dose CD34⁺ and CD3⁺ T-cells group was significantly higher than that of the low-dose group (CD34⁺: 57% vs. 29%, p = 0.009; CD3⁺: 63% vs. 18%, p < 0.001). No other clinical factors or cell subsets correlated with aGVHD incidence.ConclusionsOur analysis indicates that the CD34⁺ and CD3⁺ T-cell numbers in the allografts could be the risk factors for the development of severe aGVHD (level II–IV). Further studies should aim to optimize the critical number of CD34⁺ and CD3⁺ T-cells to reduce the risk of severe aGVHD occurrence in pediatric patients.     

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/cxC57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4+ and CD8+ T cells 7 and 21 days after donor cell transfer. The populations of CD8+CD45RBlow and CD8+CD44high cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8+ T cells leading to enhanced graft survival.     

抗原特异性CD4+CD25+T细胞对同种异体胰岛移植影响及作用机制研究

目的:探讨抗原特异性CD4+CD25+Treg细胞免疫对同种异体胰岛急性移植排斥反应的影响和机制。方法:用MACS分选供体抗原特异性CD4+CD25+Treg细胞免疫糖尿病BALB/cByJ受体小鼠，以ICR小鼠胰岛为供体行同种异体胰岛移植。观察移植后小鼠的存活时间、移植前后外周血CD4+和CD8+T细胞亚群的变化和移植物中Th1/Th2细胞因子mRNA表达水平的变化。结果:抗原特异性Treg细胞联合胰岛移植组(C组)胰岛移植物平均生存期为(34.57±17.15)d，显著长于单纯胰岛移植组(B组)的(10.6±1.82) d (P<0.01)；移植后第3天，C组外周血CD4+/CD8+的值显著低于B组(P<0.01)；C组移植物中IL-10,TGF-β mRNA表达比B组显著增强。B组移植物中IL-1β，IL-2及IFN-γ mRNA表达明显强于C组。结论:抗原特异性CD4+CD25+ Treg细胞可通过调节Th2/Th1之间的反应平衡而延长同种异体胰岛移植物的存活时间。     

Induction of bona fide regulatory T cells after liver transplantation - the potential influence of polyclonal antithymocyte globulin

T‐cell‐depleting strategies are an integral part of immunosuppressive regimens used in the hematological and solid organ transplant setting. Besides prevention of alloreactivity, treatment with rabbit antithymocyte globulin (rATG) has been related to the induction of immunoregulatory T cells (Treg) in vitro and in vivo. To investigate Treg induced by rATG, we prospectively studied the effect of rATG induction therapy in liver‐transplanted recipients in vivo (n = 28). Treg induction was further evaluated by means of Treg‐specific demethylation region (TSDR) analysis within the FOXP3 locus. Whereas no induction of CD4⁺ CD25^highCD127^? Treg could be observed by phenotypic analysis, we could demonstrate an induction of TSDR⁺ T cells within CD4⁺ T cells exclusively for rATG‐treated patients in the long‐term (day 540) compared with controls (P = NS). Moreover, although in vitro experiments confirm that rATG primarily led to a conversion of CD4⁺ CD25^? into CD4⁺ CD25⁺ T cells displaying immunosuppressive capacities, these cells cannot be classified as bona fide Treg based on their FOXP3 demethylation pattern. Consequently, the generation of Treg after rATG co‐incubation in vitro does not reflect the mechanisms of Treg induction in vivo and therefore the potential clinical relevance of these cells for transplant outcome remains to be determined.