UvA-DARE (Digital Academic Repository) Lymphocytes in the frontline: local human T cells facing the challenges of the lung environment Piet, B. Publication date 2011 Link to publication Citation for published version (APA): Piet, B. (2011). Lymphocytes in the frontline: local human T cells facing the challenges of the lung environment. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:01 Oct 2021 Chapter The organ-specific transcriptome of human lung CD8+ T cells 2 Abstract It is unknown whether human lung T cells, like other lymphocytes, recirculate or rather belong to a distinct tissue-specific population. This issue is important for understanding 2 their role in the defense against viral infection and their contribution to pathophysiology of lung diseases such as chronic obstructive pulmonary disease (COPD). By comparing G ene transcriptional profiles of blood and lung CD8+ T cells, we aimed to reveal specific + expression traits of lung CD8 T cells. Cluster analysis showed that localization of T cells had a far greater impact on mRNA expression profiles than differentiation state or inter-individual variability. Pathways guiding cellular recruitment, retention, proliferation and survival of CD8+ T cells in the lung were highly enriched for differentially expressed genes. Specifically, of chemokine receptors, chemokine receptor ligands and integrins were upregulated in lung lung T cells. Interestingly, our expression data suggested a strong translational suppression in lung CD8+ T cells compared to peripheral blood. Thus, lung CD8+ T cells have an organ- CD8 specific transcriptome, with high expression of genes that could play a role in cell migration, + T survival and retention. cells Berber Piet1,2, Perry D. Moerland3, René E. Jonkers2 and René A. W. van Lier1,4 1 Department of Experimental Immunology, 2 Department of Pulmonology and 3 Bioinformatics Laboratory, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Centre, Amsterdam, the Netherlands; 4 Division of Research, Sanquin Blood Supply Foundation, Amsterdam, the Netherlands 46 Introduction COPD is an inflammatory lung disease, characterized by a non-reversible, progressive airflow limitation, and severe morbidity. It is the 4th cause of death worldwide and due to a lack of effective therapies, the number of deaths is still increasing. Better knowledge of 2 the pathogenesis of COPD will lead to new clues for therapeutic interference. Increased G numbers of CD8+ T cells have been found in lungs of COPD patients and their presence in ene 1 lung and airways correlates with the amount of airflow limitation . It has therefore been expression hypothesized that CD8+ T cells are instrumental in the pathogenesis of COPD. However, the processes that drive this local accumulation and the pathophysiological role of CD8+ T cells are unknown. Increased lung CD8+ T cell numbers may result from enhanced recruitment, local of proliferation, increased survival, or a combination of these processes. Studies in mice have lung shown that respiratory infection is one of the main factors driving T cell recruitment to the CD8 lung 2;3. Post-infection a virus-specific memory T cell pool is maintained in the lungs that 2-5 + provides protection against re-infection . Retention and survival of these murine virus- T specific T cells is achieved via integrin-mediated mechanisms 5;6. Our previous research cells suggested that a similar virus-specific memory CD8+ T cell population is maintained in human lungs 7;8. Moreover, we demonstrated that this CD8+ T cell population differs phenotypically and functionally from its peripheral blood counterpart 7;8. Previous microarray studies using lung tissue either have compared expression profiles between total peripheral blood and lung T cells 9 or used total lung homogenates to identify gene expression differences between COPD and healthy lung tissue 10-12. The latter studies mainly identified differentially regulated genes involved in tissue remodeling and repair. We here compared gene expression profiles of paired human peripheral blood and lung CD8+ T cells to determine which genes may guide the specific (patho)physiological processes of lung CD8+ T cells. Results Location has a major impact on CD8+ T cell mRNA expression profiles We compared the gene expression profiles of non-naïve circulating CD8+ T cells with non-naïve lung CD8+ T cells. By sorting only CD45R0+ cells we prevented a bias due to the different contribution of naïve cells to the peripheral blood and lung CD8+ T cell pool 7. To distinguish between mechanisms that may play specific roles in either effector- or memory- type CD8+ T cells, we subdivided the lung CD8+ T cell populations based on the expression of CD27 13. Gene expression levels were compared to those of pooled naïve peripheral blood CD8+ T cells 14. More than 2,700 genes were significantly differentially expressed between blood and lung. Slightly more genes were differentially regulated between the CD27- lung fraction and blood than between the CD27+ lung fraction and blood. However, the vast majority of differentially regulated genes overlapped between CD27+ and CD27- lung CD8+ T cells 47 (Figure 1A). The comparison between paired CD27+ and CD27- lung CD8+ T cell samples yielded only 15 differentially regulated genes (Supplemental Table 1 shows genes with an adjusted p value < 0.1) which was surprising as CD27+ and CD27- peripheral blood CD8+ T cells are known to have distinct gene expression profiles 14. 2 Genes encoding for proteins that are expressed in peripheral blood memory CD8+ T + + G cells like CCR7, granzyme K, CD28 and IL-8 were increased in lung CD8 CD27 cells. Genes ene that are higher expressed in effector than in memory T cells such asKLRD1 (CD94), CXCR6 expression and GZMB (granzyme B) were also increased in lung CD8+CD27- cells 14;15, corroborating the validity of our microarray data. To obtain an impression of the influence of cellular location on gene expression, we performed a cluster analysis on gene expression profiles of the different samples (Figure 1B). Strikingly, location was a greater determinant for gene of expression profile than differentiation state or inter-individual variability. lung CD8 + A T 0 cells lung CD27+ vs. lung CD27- vs. 30 32 blood: 1784 blood: 1931 1454 300 445 533 lung CD27+/- vs. blood: 2732 B 0.7 0 Memory CD8+ patient 1 Effector CD8+ patient 1 Effector CD8+ patient 2 Memory CD8+ patient 2 Memory CD8+ patient 2 (duplicate) Memory CD8+ patient 3 LUNG Effector CD8+ patient 3 Pool naive CD8+ BLOOD Pool naive CD8+ (duplicate) Effector&memory CD8+ patient 3 + Effector&memory CD8 patient 2 Effector&memory CD8+ patient 1 FIGURE 1. Location has a greater impact on CD8+ T cell RNA expression profile than differentiation state or inter-individual variability. A: Venn diagram showing genes significantly and differentially regulated between blood and lung CD8+ T cells, between blood and CD27+ lung CD8+ T cells and between blood and lung CD27-CD8+ T cells. Circles show overlap in differentially regulated genes between these three comparisons, numbers indicate amount of genes. B: Hierarchical clustering of all hybridized samples. Samples above the line are lung samples, samples below the line are peripheral blood samples. From the pool of naive CD8+ T cells and the lung memory CD8+ T cells from patient 2 duplicates were hybridized. 48 FIGURE 1. A 1.0 1.0 0.5 0.5 0.0 0.0 -0.5 -0.5 Expression Expression level Expression level -1.5 -1.5 -1.0 2 - - - - - - - + + + + + + G naive naive naive blood blood blood blood blood blood blood blood blood ene lung lung 27 lung 27 lung 27 lung 27 lung 27 lung 27 lung lung 27 lung lung 27 lung lung 27 lung lung 27 lung 27 lung lung 27 expression B Cell death Protein synthesis of Cellular development Connective tissue disorders lung Hematological system development Dermatological diseases CD8 + Hematopoiesis Genetic disorder T cells Cell-mediated immune response Inflammatory disease Cellular function and maintenance Skeletal/Muscular disorders Cancer RNA post-transcriptional modification Cellular growth and proliferation Cell cycle Dermatological diseases Cell death Gene expression Cancer Cell cycle Cellular assembly and organization Immunological disease Carbohydrate metabolism Inflammatory response Cell morphology Tissue morphology Cellular growth and proliferation Genetic disorder Cellular movement FIGURE 2. ©2000-2011 Ingenuity Systems, Inc. All rights reserved FIGURE 2. Enrichment of biological processes for genes significantly up- and downregulated between blood and lung. A: Analysis of the 940 genes that were upregulated in lung CD8+ T cells (left) and analysis of the 1053 genes that were downregulated in lung CD8+ T cells (right) compared to peripheral blood CD8+ T cells. Naive = pool of naive CD8+ T cells sorted from 5 healthy donors, blood CD45R0+ = 3 separate fractions of CD45R0+CD8+ T cells sorted from peripheral blood from 3 patients and lung CD45R0+CD27+/- = paired lung fractions of CD27+ and CD27- CD45R0+CD8+ T cells. B: Biological function enrichment profile of upregulated (left) and downregulated genes (right). Analysis performed with Ingenuity software, -log (p-value) is shown.