T suppressor lymphocytes reverse ongoing acute allograft rejection

(LEW X BN)F1 cardiac allografts are rejected within 8 days in untreated LEW recipients. At the critical time point of 5 days after transplantation, the obviously rejecting grafts are enlarged and maximally infiltrated by host cells as shown by 111In-labeled lymphocyte tracer studies. However, when such hearts were retransplanted back to naive (LEW X BN)F1 secondary hosts, they survive indefinitely, showing that even late rejection is reversible in the absence of sustained host immunological drive. Attempts were then made to abrogate this advanced immune responsiveness using Cyclosporine (CsA). CsA therapy (15 mg/kg/day for 7 days) starting from day 5 produced indefinite graft survival, similar as if initiated at the time of operation. Addition of exogenous IL-2, which drives the proliferation of Tc, could not reverse this effect. Serial changes in phenotype of lymphocyte subpopulations infiltrating both acutely rejecting and indefinitely functioning cardiac allografts in unmodified and CsA treated hosts, respectively, were then studied. Ratio of Th:Tc/s cells in acutely rejecting grafts was 1.6 by day 3; it inverted abruptly to 0.7 by day 5-6, suggesting predominance of Tc/s during the later stages of allograft rejection. Similarly, treatment with CsA produced a transient depression of Th, with recovery of original Th:Tc/s ratio during the next 2-3 weeks. Adoptive transfer experiments were then performed to investigate the functional significance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of endothelial cells in allograft rejection

Endothelial cells (EC) are crucially involved in allograft rejection. They are prime targets of alloreactivity but also key players in the recruitment and extravasation of immune cells. These mechanisms also become clear in allograft biopsies with antibody-mediated complement deposition on EC and associated intracapillary accumulation of immune cells. HLA molecules are the most prominent targets of alloantibodies in AB0 compatible transplantation. Clinically relevant antibodies against other antigens such as MICA (MHC class I-related chain A) or the angiotensin II Type-1 receptor could also be convincingly demonstrated. The lack of generally available diagnostic tests for such non-HLA antibodies hampers their introduction into clinical practice. Alloantibodies undoubtedly cause allograft rejection. However, our knowledge of the molecular mechanisms underlying graft dysfunction in antibody-mediated rejection (AMR) is still fragmentary. Activation of EC by anti-endothelial cell antibodies was demonstrated in several experimental systems. Recent animal studies employing immune cell deficient transplant recipients or in-vitro assays, however, failed to demonstrate an immediate response of EC upon antibody binding and complement activation. It might therefore be considered that direct antibody- or complement-mediated EC damage is not necessarily the leading event in acute AMR. Antibody- and/or complement-induced recruitment of immune cells might rather be of crucial importance at least in the early phases of AMR.     

NKT cells: T lymphocytes with innate effector functions

Natural killer T (NKT) cells are innate-like T lymphocytes that recognize glycolipid antigens in the context of the MHC class I-related glycoprotein CD1d. Recent studies have identified multiple ways in which NKT cells can become activated during microbial infection. Mechanisms of CD1d-restricted antigen presentation are being unraveled, and a surprising connection has been made to proteins that control lipid metabolism and atherosclerosis. It appears that several microorganisms have developed strategies to interfere with the CD1d antigen-presentation pathway. New studies have also provided important insight into the mechanisms that control effector cell differentiation of NKT cells and have revealed specialized functions of distinct NKT cell subsets. Finally, there is continued enthusiasm for the development of NKT cell-based therapies of human diseases.     

Are B lymphocytes involved in allograft/tumor rejection and GVH?

Several earlier observations by us and other investigators led us to propose a thesis that B lymphocytes, when activated by a virus, allo-antigen or bacterial protein would enter into effector arm of the immune response and cause allograft and tumor rejection or GVH-type of lesion in immunosuppressed animals. These effector cells may act directly in the process by contact with other cells or liberating cytokines. Such an hypothesis needs further support from experiments involving cancer and transplantation patients' B cells.     

Renal allograft rejection: possible involvement of lymphokine-activated killer cells

Human renal allograft tissue was recovered at transplant nephrectomy from three patients with irreversible loss of graft function. This tissue was disaggregated and separated into two fractions on the basis of particle size. Fraction 1 contained glomeruli and developed a mixed outgrowth containing adherent epithelial and mesangial cells after a limited period of culture. Fraction 2 contained fragments of renal tubules and produced monolayers of tubular epithelial cells during culture. A population of lymphoid cells was observed to grow from the primary disaggregate into medium supplemented with recombinant human interleukin-2 (IL-2). After culture for 5 days these lymphoid cells were predominantly CD3-positive and carried both class II major histocompatibility antigens (MHC) and the CD25 IL-2 receptor. Culture of peripheral blood-derived mononuclear cells with IL-2 caused the generation of lymphokine-activated killer (LAK) cells; these cells were able to lyse both glomerular and tubular cells grown from nephrectomy tissue without showing MHC antigen restriction. The lymphoid cells grown from renal allograft tissue showed a similar lytic potential for both renal cells prepared from the same nephrectomy specimen and from third party renal tissue. It is possible that any LAK cells formed within a renal allograft by the action of IL-2 may contribute to the tissue destruction observed during graft rejection.     

Molecular case report: IgVH analysis in acute humoral and cellular liver allograft rejection suggests a selected accumulation of effector B cells and plasma cells

Acute cellular (CLR) and humoral liver allograft rejection (HLR) are the most important immunological obstacles to successful liver transplantation. In HLR, serum antibodies play the central pathogenetic role. In CLR, CD3+ T lymphocytes drive the destructive immune response. Although CLR and HLR show different clinical symptoms and can be kept apart in most cases, they share histomorphological similarities. In CLR, hepatic B lymphocytes and plasma cells as well as B-cell-activating cytokines have recently been described, indicating that, in addition to T cells, antibody-mediated mechanisms might be involved. To analyze the impact of hepatic B cells in CLR and HLR, the immunoglobulin (Ig) variable (V)-region gene repertoire was determined from tissue of one case of CLR and one case of HLR. Complement deposits and lymphocytic infiltrate were determined using immunohistochemistry. T cells, B lymphocytes and plasma cells could be detected in both cases, whereas C3c and C4d deposits could only be demonstrated in the HLR case. The molecular analysis of 63 V-region genes showed that B cells in both allografts expressed selected V-gene repertoires. All sequences differed from the putative germline sequences by multiple somatic mutations. This suggests a clonal expansion of selected effector B cells in the portal tracts of liver allografts. Locally accumulated B cells and their antibodies might be involved in IgG-mediated complement activation in CLR and HLR.     

Mast cells and c-Kit expression in liver allograft rejection

AIMS : The pathogenesis of rejection following liver transplantation is not fully understood. It has been postulated that mast cells may play a role in acute and chronic rejection of a number of other solid organ grafts. The aim of this study was to assess the possible role of mast cells and c-Kit+ cells in acute and chronic liver allograft rejection. METHODS AND RESULTS : Biopsy specimens from (i) 'time zero' grafts with a minimal degree of perfusion injury (controls), (ii) transplanted livers with different grades of acute rejection, and (iii) transplanted livers with end-stage chronic rejection, were stained immunohistochemically using monoclonal anti-mast cell tryptase and polyclonal anti-c-Kit antibodies. Tryptase- and c-Kit-positive cell densities were assessed by image analysis. Tryptase-positive mast cell densities (P<0.001) were strongly correlated with acute liver allograft rejection grades and chronic liver allograft rejection. Furthermore, a similarly strong relationship was found between c-Kit+ cell densities and increasing rejection grade (P<0.001). CONCLUSIONS : Tryptase- and c-Kit-positive mast cells form part of the inflammatory infiltrate in both acute and chronic liver allograft rejection, and may be important effector cells in these processes.     

Immunological monitoring and early prediction of rejection in renal allograft recipients

Graft rejection remains the major problem complicating renal allograft transplantation. A reliable posttransplant predictor of impending rejection will be valuable to help maintain better graft function. We monitored 47 patients with end-stage renal disease treated by renal allograft starting 1 day pretransplantation and continuing for up to 90 days postgrafting. Peripheral blood mononuclear cells (PBMC) from both patients and 71 healthy subjects were compared for: (1) DNA synthesis in T and B lymphocytes in response to mitogens; (2) interleukin-2 (IL-2) production; (3) natural killer (NK) and antibody-dependent cell-mediated cytotoxic (ADCC) activities; (4) induced augmentation of NK and ADCC activities by the biological response modifiers (BRM), lymphoblastoid interferon, recombinant alpha-2-interferon, gamma-interferon and recombinant IL-2. During the 2 weeks preceding rejection we found lower than normal levels of IL-2 production (p less than 0.0005) and DNA synthesis (p less than 0.01) in concanavalin A-stimulated PBMC. IL-2 yield reached its lowest level on the day of rejection and increased sharply the following week after antirejection therapy was started. Mitogen-stimulated DNA synthesis rose in parallel with increasing levels of IL-2 production. Both NK and ADCC activities increased during rejection (p less than 0.05). The ADCC response to BRM activation measured during the first 2 weeks postgrafting was found to correlate with the stability of the graft. Recipients whose graft function remained stable had a minimal ADCC response to BRM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolated lymphocytes can restore allograft rejection capacity of early-thymectomized Xenopus

In the clawed toad, Xenopus laevis, thymectomy at 4 days of age abrogates the allograft rejection capacity. Restoration of allograft immunity to approximately normal levels was achieved after injecting 1 × 10⁷ thymic or 2 × 10⁶ splenic lymphocytes from histocompatible toads. Lymphocytes from peripheral blood were as effective as thymocytes. The degree of restoration was dependent upon the number of injected thymocytes. Histoincompatible thymocytes were also effective in restoration, although to a lesser degree. In all restored toads, repopulation of lymphocytes was evident in the spleen.     

Tregs调节小鼠肝癌局部引流淋巴结内免疫效应细胞的功能

目的:探讨肿瘤引流淋巴结(TDLNs)内调节性T细胞(Tregs)对局部免疫效应细胞的调节作用。方法:建立小鼠肝癌TDLNs模型,通过免疫组织化学染色和流式细胞仪检测TDLNs内Foxp3+Tregs和CD4+及CD8+T细胞的数量。实时定量PCR测定Foxp3mRNA表达水平。应用酶联免疫斑点法(ELISPOT)检测TDLNs内CD8+T细胞分泌IFN-γ的功能。结果:TDLNs内Tregs和效应性T细胞均明显扩增,Tregs弥散分布于CD8+T细胞聚居区。TDLNs内Foxp3mRNA表达水平显著高于同一接种肿瘤小鼠腹股沟淋巴结(P0.01)和脾脏(P0.01)。Tregs趋向于在TDLNs内聚集,而非其它外周淋巴结位点。荷瘤小鼠的脾脏Foxp3mRNA表达明显高于注射LPS小鼠脾脏。Tregs抑制TDLNs内已初始化的CD8+T细胞分泌IFN-γ的功能,经anti-CD3刺激激活后,CD8+T细胞分泌IFN-γ的功能可恢复。结论:TDLNs内Tregs通过调控CD8+T细胞功能而发挥重要作用,清除Tregs是发挥特异性肿瘤免疫治疗的关键。     

Apoptosis of bile duct epithelial cells in hepatic allograft rejection

Liver biopsy remains the 'gold standard' for monitoring rejection in liver transplant patients. Portal inflammation, bile duct damage and endothelialitis are recognized features of hepatic allograft rejection. The pathogenesis of the bile duct injury during rejection, however, remains unclear. To define the mechanism of bile duct damage, we studied the light- and electronmicroscopic appearance of hepatic tissue from selected patients in whom allograft failure was solely due to rejection. Of the 25 orthotopic liver transplant rejection cases examined, 17 were mild, seven were moderate and one was severe rejection. Light microscopy examination of the damaged bile duct epithelium revealed evidence of apoptosis which was confirmed by electronmicroscopy. Furthermore, there appeared to be a positive correlation between the grade of rejection and the number of apoptotic cells. Also included in the study were 13 cases of chronic active hepatitis and 10 normal livers which showed the least apoptotic cells. We conclude that the identification of apoptotic cells in damaged bile ducts in allograft biopsies might be helpful in the diagnosis of rejection and in assessment of the severity of rejection.     

同种异体神经干细胞脑内移植免疫排斥反应的实验研究

目的观察帕金森病(PD)模型大鼠同种异体神经干细胞(NSC)脑内移植是否存在免疫排斥反应。方法取E14.5d SD胚鼠腹侧中脑组织进行分离、培养、扩增,经Brdu、Nestin免疫组化染色鉴定确认为NSC后,借助脑立体定位仪移植到SD大鼠PD模型的纹状体内,分别于10、21、35、60d时检测其旋转行为后分批处死,再行HE、CD4、CD8、酪氨酸羟化酶(TH)和MHC-Ⅱ抗原免疫组化染色。结果移植组10d时,CD4、CD8、、MHC-Ⅱ少量表达,以21d时较为明显,35d时明显减少,60d消失;10d、21d时移植区未见明显TH阳性神经元,35d后数量显著增加。阳性对照组在10d、21d时见少量CD4、CD8、和MHC-Ⅱ阳性细胞,35d时消失,各时间点都未见TH阳性神经元。阴性对照组在各时间点都未见TH阳性神经元和CD4、CD8、MHC-Ⅱ阳性细胞。PD模型移植组旋转行为35d时出现改善。结论神经干细胞脑内移植未见明显免疫排斥反应。     

外周血淋巴细胞穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应的早期诊断价值

探讨外周血淋巴细胞(peripheral blood lymphocyte,PBL)穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应(AR)的早期诊断价值。采用RT-PCR方法动态检测67例肾移植AR患者外周血淋巴细胞穿孔素和颗粒酶B mRNA的表达水平,同时检测所有患者肌酐(Cr)表达水平情况,比较各时间点穿孔素、颗粒酶B mRNA及Cr表达水平,并探讨穿孔素、颗粒酶B与AR的关系。移植后1d与移植前1d比较,PBL穿孔素、颗粒酶B mRNA表达水平比较无明显差异性(P>0.05),Cr表达水平明显下降(P<0.05);AR前3d较之前各时间点比较,PBL穿孔素、颗粒酶B mRNA表达水平明显上升(P<0.05),而Cr表达水平明显下降(P<0.05);AR后1d时PBL穿孔素、颗粒酶B mRNA表达水平达到最高峰,随后逐渐下降(P<0.05),而AR后5d时Cr表达水平达到最高峰,随后逐渐下降(P<0.05);PBL穿孔素、颗粒酶B mRNA表达水平升高时间比Cr早4d,AR患者经MP/OKT3冲击治疗后,PBL穿孔素和颗粒酶B mRNA表达水平逐渐降低至原有基础水平。PBL穿孔素和颗粒酶B mRNA在AR早期诊断和抗排斥反应疗效评估等方面具有一定的临床价值。     

IL-4 expression delays eosinophil-independent vasculopathy and fibrosis during allograft rejection in the mouse

Transplant vasculopathy in the mouse is thought to be dependent on IL-4 and mediated by IL-5 and eosinophils, whereas in the rat and human systems, IL-4 is associated with the absence of transplant vasculopathy and down-regulation of a Th1-type response. In this study we tested the possibility that the apparent difference in the role of IL-4 in transplant vasculopathy is related to protocol differences rather than to the species being studied. Using a protocol that closely resembles that used in rat and human studies, we developed a model of transplant vasculopathy in the mouse that is associated with Th1-type cytokines and independent of IL-5 and eosinophil infiltration. In this model IL-4 promotes a significant delay in vasculopathy in the graft (P = 0.04) and a decrease in the incidence of allograft rejection (P = 0.02). The data suggest that the role of IL-4 in transplant vasculopathy can be controlled by the protocol used to treat the transplant recipient.     

Evidence for the early involvement of interleukin 17 in human and experimental renal allograft rejection

Inflammatory processes can stimulate renal epithelial cells to release cytokines, chemoattractants and matrix proteins into the interstitium, thus contributing to interstitial injury during acute allograft rejection. To test the role of interleukin 17 (IL-17) in this process, cultured human renal epithelial cells (hRECs) were first established and treated with or without human IL-17 (hIL-17) for 2, 4, 8 and 10 h in vitro. Significant elevations of IL-6 and IL-8 levels were noted in the supernatants in a dose-dependent and time-dependent manner, as also for IL-6 mRNA expression. Secondly, using a rat acute allograft rejection model, the correlation between IL-17 expression and histopathological changes was serially studied. The results demonstrated that increased expression of IL-17 protein on infiltrating mononuclear cells (MNCs) was detectable on day 2. This corresponds to the borderline change of acute rejection according to the Banff classification, and it increased progressively to day 5. Serial study of IL-6, IL-8 and IL-17 mRNA expression of the renal allograft confirmed IL-17 mRNA expression in the allograft early on post-transplant day 2, whereas IL-6 and IL-8 expression started on day 3. Thirdly, IL-17 expression was observed in human renal allograft and urinary sediment. IL-17 protein expression was found in human subclinical (borderline) rejection renal allograft biopsy tissue and none in biopsy tissue not showing any evidence of rejection. There was also a 100% detectable rate of IL-17 mRNA expression in the MNCs of urinary sediment of patients with subclinical borderline rejection. These results demonstrate that hRECs exposed to IL-17 can produce inflammatory mediators with the potential to stimulate early alloimmune responses, which may also serve to give warning of acute renal allograft rejection.     

The frequency of effector cells in populations containing cytotoxic T lymphocytes

A new approach for the direct enumeration of effector cells in populations containing cytotoxic T lymphocytes is described. Stable lymphocyte-target cell conjugates are formed during centrigugation at room temperature of mixtures containg cytotoxic lymphocytes and target tumor cells. Various types of lymphocyte-target cell conjugates can be formed, the composition of the conjugate being dependent upon the ratio of lymphocytes to target cells during the conjugation process. The number of conjugates formed is highest at equal lymphocyte and target cell concentrations. At least 35% of the BALB/c anti-EL4 peritoneal exudate lymphoid cells can conjugate to target cells and the majority of the conjugated lymphocytes are also killer cells. The variation in cytolytic activity of lymphoid cells with time following immunization and the differences in the lytic activity of immune lymphoid cells from various lymphoid organs are due to differences in the actual number of effector cells capable of conjugating with target cells.