Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy

Transplant rejection involves a coordinated attack of the innate and the adaptive immune systems of the host. To investigate this dynamic process and the contributions of both donor and host cells, we developed an ear skin graft model suitable for intravital imaging. We found that donor dermal dendritic cells (DCs) migrated rapidly from the graft and were replaced by host CD11b(+) mononuclear cells. The infiltrating host cells captured donor antigen, reached the draining lymph node and cross-primed graft-reactive CD8(+) T cells. Furthermore, we defined the mechanisms by which host T cells target graft cells. We found that primed T cells entered the graft from the surrounding tissue and localized selectively at the dermis-epidermis junction. Later, CD8(+) T cells disseminated throughout the graft and many became arrested. These results provide insights into the antigen presentation pathway and the stepwise progression of CD8(+) T cell activity, thereby offering a framework for evaluating how immunotherapy might abrogate the key steps in allograft rejection.     

The innate immune system in allograft rejection and tolerance

As T cells alone are both necessary and sufficient for the rejection of virtually all allogeneic tissues, much of transplantation immunology has focused on cells of the adaptive immune system. During the past decade, advances in our understanding of innate responses to pathogen-associated molecules have spurred a "rediscovery" of innate immunity. Fueled by this, an increasing body of literature has emerged in which the role of the innate immune system in allograft rejection and tolerance has been examined more closely. This review will give an overview of recent studies and emerging concepts of how the cellular components of the innate immune system participate in the immune response to solid organ transplantation. These important studies highlight the complex interplay between diverse cells of the immune response and provide the basis for optimal strategies of tolerance induction.     

The innate NK cells, allograft rejection, and a key role for IL-15

Transplant rejection is mediated primarily by adaptive immune cells such as T cells and B cells. The T and B cells are also responsible for the specificity and memory of the rejection response. However, destruction of allografts involves many other cell types including cells in the innate immune system. As the innate immune cells do not express germline-encoded cell surface receptors that directly recognize foreign Ags, these cells are thought to be recruited by T cells to participate in the rejection response. In this study, we examined the alloreactivity of the innate NK cells in Rag(-/-) mice using a stringent skin transplant model and found that NK cells at a resting state readily reject allogeneic cells, but not the skin allografts. We also found that IL-15, when preconjugated to its high affinity IL-15Ralpha-chain, is remarkably potent in stimulating NK cells in vivo, and NK cells stimulated by IL-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells. Furthermore, NK cell-mediated graft rejection does not show features of memory responses. Our data demonstrate that NK cells are potent alloreactive cells when fully activated and differentiated under certain conditions. This finding may have important clinical implications in models of transplantation and autoimmunity.     

Control of innate and adaptive immunity by the inflammasome

The importance of innate immunity lies not only in directly confronting pathogenic and non-pathogenic insults but also in instructing the development of an efficient adaptive immune response. The Nlrp3 inflammasome provides a platform for the activation of caspase-1 with the subsequent processing and secretion of IL-1 family members. Given the importance of IL-1 in a variety of inflammatory diseases, understanding the role of the Nlrp3 inflammasome in the initiation of innate and adaptive immune responses cannot be overstated. This review examines recent advances in inflammasome biology with an emphasis on its roles in sterile inflammation and triggering of adaptive immune responses.     

Analysis of cytokine dynamics in corneal allograft rejection

Motivated by the discovery of oscillations in tumour necrosis factor-alpha (TNF-alpha) concentration in the aqueous humour of rabbits undergoing corneal allograft rejection, a simple mathematical model is developed for the regulation of TNF-alpha, which incorporates both negative feedback and amplification pathways. Mathematical analysis reveals a surprisingly rich behavioural repertoire for this simple cytokine pathway, including excitability, threshold behaviour, hysteresis, oscillations and bistability. This suggests new possibilities for experimental demonstration, and reveals the potential contributions of nonlinear dynamics to understanding cytokine regulation.     

Evasion of innate and adaptive immunity by flaviviruses

After a virus infects an animal, antiviral responses are generated that attempt to prevent dissemination. Interferons, antibody, complement, T and natural killer cells all contribute to the control and eradication of viral infections. Most flaviviruses, with the exception of some of the encephalitic viruses, cause acute disease and do not establish persistent infection. The outcome of flavivirus infection in an animal is determined by a balance between the speed of viral replication and spread, and the immune system response. Although many of the mechanistic details require further elucidation, flaviviruses have evolved specific tactics to evade the innate and adaptive immune response. A more thorough understanding of these principles could lead to improved models for viral pathogenesis and to strategies for the development of novel antiviral agents.     

Shaping of adaptive immunity by innate interactions

In inflamed tissues, the reciprocal interaction between Natural Killer (NK) cells and Dendritic Cells (DC) results in a potent activating cross talk that leads to DC maturation and NK cell activation with acquisition of NK-mediated cytotoxicity against immature DC (iDC). We focused our studies on NK-mediated killing of monocyte-derived iDC and we provided evidence that NK cells that express CD94/NKG2A but not killer Ig-like receptors (KIR) are able to kill autologous iDC. Indeed HLA-E (i.e. the cellular ligand of CD94/NKG2A) is sharply reduced in iDC, whereas it is partially recovered in mDC. The latter are lysed only by a small fraction of NK clones characterized by low levels of CD94/NKG2A expression. Another NK receptor, whose surface density is crucial for the ability to kill iDC, is represented by NKp30, a member of the NCR (Natural Cytotoxicity Receptor) family. We showed that transforming growth factor beta1 (TGFbeta1) treatment results in specific downregulation of NKp30 expression. This effect profoundly inhibits the NK-mediated killing of DC suggesting a possible mechanism by which TGFbeta1-producing DC may acquire resistance to the NK-mediated attack.     

多杀菌素生物合成基因簇启动子探测和转录时序

多杀菌素是对农业虫害防治及粮食仓储安全均具有重大意义的农用抗生素.为了深入揭示刺糖多孢菌合成多杀菌素的调控特点,首先通过建立基于报告基因的启动子探测技术,探测了多杀菌素生物合成基因簇的9个启动子活性.并进一步通过荧光定量PCR,分析了这9个基因和不在基因簇内的负责糖基前体供应和鼠李糖合成的4个基因的转录时序,结果表明多杀菌素生物合成基因簇内的9个基因在菌体生长进入稳定期时有较高的转录,这和发酵液中此时开始大量积累多杀菌素一致;同时还发现,簇外的4个与糖基供应相关的基因和基因簇内基因的转录时序不同,它们在菌体生长对数期有较高的转录活性,这暗示多杀菌素聚酮链的合成速率和参与后修饰的糖基底物供应的最优化匹配有可能是提高生物合成多杀菌素的前提和关键.     

Detection of renal allograft rejection by computer.

A computer program incorporating an adaptation of a statistical method, the multiprocess Kalman filter, was used to detect changes in trends of plasma creatinine and urea concentrations. In 28 recipients of renal allografts a definite deterioration in renal function was identified retrospectively on 32 occasions by an experienced renal physician independently of the statistical analysis. The computer identified 31 of these 32 episodes using creatinine and urea results, and 29 using creatinine alone. Dysfunction was identified by the computer significantly earlier (p less than 0.05) than by the clinician and a median of one day earlier (p less than 0.02) than treatment was actually initiated. The computer identified dysfunction on 11 out of 1259 days when the clinician did not suspect rejection. These 11 episodes may have had a pathological importance, though no clinical diagnosis was made. This computer method is useful for immediate analysis of incoming results and for timing events either prospectively or retrospectively.     

Genetic analysis of innate immunity: TIR adapter proteins in innate and adaptive immune responses

The innate immune system senses pathogens largely through signals initiated by a collection of phylogenetically related proteins known as "Toll-like receptors" (TLRs), of which 10 representatives are encoded in the human genome. Our understanding of the sensing role played by the TLRs began with the positional cloning of a spontaneous mutation (Lps(d)) in the gene encoding the mammalian lipopolysaccharide (LPS) receptor. Other key innate immunity proteins have been disclosed by germline mutagenesis, and are discussed in the present review.     

Characterization of acute renal allograft rejection by proteomic analysis of renal tissue in rat

Rapid and reliable biomarkers of renal allograft rejection have not been available. This study aimed to investigate biomarkers in renal allograft tissue using proteomic analysis. Orthotopic kidney transplantations were performed using Fisher (F344) or Lewis rats as donors and Lewis rats as recipients. Syngenic control group (Group I) constituted F344-to-F344 orthotopic kidney allo-transplantations (n = 8); and allogenic group (Group II) consisted of F344-to-Lewis orthotopic kidney allo-transplantations (n = 8). Renal tissues were harvested 7 days after transplantation. Samples were analyzed using 2-D electrophoresis and matrix assisted laser desorption ionization-time of flight mass spectrometry. 6 differentially expressed proteins were identified between allogenic group and syngenic control group. A rat model of acute renal allograft rejection was successfully set up. Differentially expressed proteins in renal allograft tissue of rat were detected using proteomic analysis and might serve as novel diagnostic and therapeutic targets in human. Quantitative proteomics, using MALDL-TOF-MS methodology has the potential to provide a profiling and a deeper understanding of acute renal rejection.     

Analysis of chronic lung transplant rejection by MALDI-TOF profiles of bronchoalveolar lavage fluid

While lung transplant is an effective therapy for advanced lung disease, chronic allograph rejection remains a primary basis for lower survival rates than those for other solid organ transplants. This study used carefully controlled Zip-Tip extraction of bronchoalveolar lavage fluid (BALF) followed by MALDI-TOF MS to identify biomarkers of chronic lung transplant rejection. Many differences were observed between controls, those who did not develop chronic rejection within 100 months, and patients who had developed chronic rejection, diagnosed as bronchiolitis obliterans syndrome (BOS). Intensity ratios of peaks within the same MALDI-TOF profile were used to quantify the result. One of the best identifiers of BOS was a lowered ratio of clara cell protein (CCP m/z = 15,835) to lysozyme (m/z = 14,700), which gave 94% specificity and 74% sensitivity for diagnosis. Furthermore, low values for CCP/Lysozyme (<0.3) were observed in 66% of samples taken at 1 to 15 months prior to the diagnosis of BOS. Many other components of the profile gave similar or better outcomes for diagnosis but tended to be less valuable for the prediction of future disease. Overall, this study demonstrated the feasibility of this approach for the detection of disease biomarkers.     

A proteomic analysis of allograft rejection in rats after liver transplantation

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft rejection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation. These authors contributed equally to this work Supported by the Key Basic Research and Development Program of China (Grant No. 2003CB515506), and in part by the Chinese Human Liver Proteome Project (CNHLPP) (Grant No. 2004BA711A18)     

A proteomic analysis of allograft rejection in rats after liver transplantation

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft rejection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation.     

A proteomic analysis of allograft rejection in rats after liver transplantation

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft re- jection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation.