Yurong Liang

Analysis of the Innate and Adaptive Phases of Allograft Rejection by Cluster Analysis of Transcriptional Profiles

Both clinical and experimental observations suggest that allograft rejection is a complex process with multiple components that are, at least partially, functionally redundant. Studies using graft recipients deficient in various genes including chemokines, cytokines, and other immune-associated genes frequently produce a phenotype of delayed, but not indefinitely prevented, rejection. Only a small subset of genetic deletions (for example, TCRα or β, MHC I and II, B7-1 and B7-2, and recombinase-activating gene) permit permanent graft acceptance suggesting that rejection is orchestrated by a complex network of interrelated inflammatory and immune responses. To investigate this complex process, we have used oligonucleotide microarrays to generate quantitative mRNA expression profiles following transplantation. Patterns of gene expression were confirmed with real-time PCR data. Hierarchical clustering algorithms clearly differentiated the early and late phases of rejection. Self-organizing maps identified clusters of coordinately regulated genes. Genes up-regulated during the early phase included genes with prior biological functions associated with ischemia, injury, and Ag-independent innate immunity, whereas genes up-regulated in the late phase were enriched for genes associated with adaptive immunity.

Graft produced interleukin-6 functions as a danger signal and promotes rejection after transplantation

Background. Interleukin (IL)-6 is a pleiotropic cytokine that functions in both the innate and adaptive immune responses. However, the role of IL-6 in allograft rejection remains poorly understood. Methods. In this study, we demonstrate a critical role for graft-produced IL-6 in allograft rejection in a murine model of cardiac allograft transplantation. Results. The results show that IL-6-deficient grafts transplanted into allogeneic wild-type recipients have significantly prolonged survival, approximately three times the survival time of wild-type controls. In contrast, allogeneic cardiac transplants into IL-6-deficient recipients do not have prolonged graft survival, indicating that donor graft cells are the relevant source of IL-6. Our investigation of potential mechanisms shows that graft-produced IL-6 promotes the activation of peripheral CD4 and CD8 T cells. Furthermore, we show that IL-6 deficiency prolongs graft survival only in the presence of CD25+ T cells that have a phenotype consistent with regulatory T cells. Interestingly, IL-6 production by the graft is triggered by antigen-independent innate immune mechanisms. Conclusions. Thus, our results suggest the paradigm that graft rejection versus tolerance is determined by a balance between the activation of effector T cells versus immune suppression by regulatory T cells, and that after transplantation, IL-6 functions as a systemic danger signal that overcomes constitutive immune suppression mediated by regulatory T cells and promotes the activation of effector T cells.

Analysis of the major histocompatibility complex in graft rejection revisited by gene expression profiles

Background. The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains incompletely understood. The important role of foreign peptides in the alloimmune response was recently recognized. Methods. We performed a comparative study of the functions of minor antigens Class I, Class II, and CD1 in murine cardiac allograft rejection by investigating the expression of a large panel of immune and inflammatory genes. To investigate the role of MHC Class II and I, our protocol analyzed allograft recipients deficient in MHC Class II and b2 microglobulin (b2-M), a critical component of the Class I heterodimer. We also included CD1 deficient recipients to differentiate effects in the &bgr;2-M deficient strain due to CD1 deficiency versus the combined inactivation of CD1 and Class I. The serum cytokines tumor necrosis factor (TNF)-&agr;, interleukin (IL)-6, interferon (IFN)-&ggr; and IL-1&bgr; were evaluated posttransplant by ELISA. The intragraft expression of 55 chemokines, chemokine receptors, and CD markers were measured by ribonuclease protection assay. The data were analyzed through hierarchical clustering dendrograms and self-organizing maps. Results. The analysis indicates that each gene deficiency induces both the upregulation and the downregulation of distinct subsets of genes and that similar kinetics of rejection can be attributed to different molecular mechanisms. Conclusions. The study provides novel insights into the role of classical and non-classical MHC molecules in graft rejection.

Modulation of gene expression by alloimmune networks following murine heart transplantation

The objective of our study was to analyze gene expression profiles in a complex in vivo model of solid organ transplantation, and to investigate the effects of single-gene deletions on alloimmunity. Using algorithms to generate dendrograms and self-organizing maps, we differentiated the alloimmune profiles of 16 transgenic knockout mouse strains, and identified subsets of genes that correlate with the duration of graft survival and provide candidates for prognostic and diagnostic indicators following transplantation in our model system.

Analysis of immunoglobulin and T-cell receptor gene deficiency in graft rejection by gene expression profiles

Background. Lymphocyte antigen receptors are critical for allograft rejection, but their precise involvement is only partly understood. Some members of the immunoglobulin superfamily (e.g., &agr;&bgr; T-cell receptors [TCR]) are known to be crucial for acute allograft rejection, but the role of other members remains poorly defined. Methods. In this study, the authors analyzed two poorly understood receptors, TCR&ggr;&dgr; and B-cell receptors (BCR), in allograft rejection in a murine model of cardiac transplantation. Using ribonuclease protection assays, their approach was a comprehensive analysis of the expression of immune and inflammatory genes. The mice included those with deficiencies of &ggr;&dgr; TCR or BCR and alymphoid (recombination activating gene [RAG] knockout) mice. Because the RAG mice lack all TCR and BCR, they provide a baseline with which to compare the effects of TCR&ggr;&dgr; and BCR deficiency. Results. Graft survival was extended in the TCR&ggr;&dgr;- and BCR-deficient mice. Furthermore, the authors were able to identify groups of genes modulated by specific receptors. Five and six genes were expressed at lower levels in the BCR- and TCR&ggr;&dgr;-deficient groups, respectively. The authors also found eight genes that were at higher levels in the TCR&ggr;&dgr;-deficient group, suggesting that these receptors have both pro- and antirejection effects. Conclusions. Overall, the authors’ study shows that the individual regulatory effects of these different lymphocyte antigen receptors are distinct. In addition, several genes are identified that are candidates as necessary for rejection. This has crucial implications for developing clinical therapies that target specific mechanisms for prolonging graft survival.

Analysis of cytokine functions in graft rejection by gene expression profiles.

Background. The function of interferon (IFN)&ggr; in the regulation of the immune response after allogeneic transplantation is still poorly understood. Previous studies have suggested that IFN&ggr; can promote rejection and be important in tolerance induction. Methods. To analyze the various IFN&ggr;-dependent functions in terms of T helpers 1 and 2 responses during rejection, we investigated mice deficient in the transcription factors (signal transducer of activated T cells [STAT]4 and 6) and IFN&ggr; in fully major histocompatibility complex-mismatched vascularized cardiac transplants. Serum levels of the cytokines tumor necrosis factor-&agr;, IFN&ggr;, and interleukin (IL)-1&bgr; were evaluated by enzyme-linked immunosorbent assay, and the graft-infiltrating cells were examined by immunohistochemical staining. To analyze a large panel of immune parameters, we determined the expression of chemokines, chemokine receptors, and clusters of differentiation markers by RNAase protection assays. The data were analyzed with algorithms that generated hierarchic clustering dendrograms. Also, the expression profiles of individual genes were determined with self-organizing maps. Results. Our data show that both the STAT4- and STAT6-deficient groups have statistically prolonged graft survival (P <0.04 and P <0.01). Despite the absence of prolongation of graft survival in the IFN&ggr;-deficient group, our analysis of variance data show that more genes (18) were modulated in the IFN&ggr;-deficient group compared with the other two STAT4- and STAT6-deficient groups (five each). Conclusions. Our results indicate that IFN&ggr; plays a distinct role in the modulation of gene expression that includes STAT4-independent mechanisms. Our study identifies eight genes (IL-1&bgr;, IL-1RA, macrophage inflammatory protein-1&bgr;, monocyte chemoattractant protein-1, CC-chemokine receptor (CCR)-1, CCR2, CCR5, and F4/80) that are highly expressed in all of our experimental groups. Thus, these genes become candidates for essential functions during rejection.

Microarray analysis of gene expression in murine cardiac graft infiltrating cells

Microarray technology can rapidly generate large databases of gene expression profiles. Our laboratory has applied these techniques to analyze differential gene expression in cardiac tissue and cells based on mouse heart transplantation. We have analyzed the different gene expression profiles such as stress or injury including ischemia following transplantation. We also have investigated the role of infiltrating inflammatory cells during graft rejection by purifying subsets of infiltrating cells using GFP transgenic mice and detailed all technical experiences and issues. The purpose of this study is to assist researchers to simplify the process of analyzing large database using microarray technology.