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Pathophysiology of intestinal intraepithelial lymphocytes: Functional heterogeneity and plasticity of CD8aa TCRab T cells

English title Pathophysiology of intestinal intraepithelial lymphocytes: Functional heterogeneity and plasticity of CD8aa TCRab T cells
Applicant Müller Christoph
Number 138392
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pathologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Immunology, Immunopathology
Start/End 01.11.2011 - 31.10.2016
Approved amount 623'000.00
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Keywords (5)

intestinal immunity; intraepithelial lymphocytes; intestinal microbiota; autoreactivity; colitis

Lay Summary (English)

Lead
Lay summary

The intestinal epithelium represents the interface between the intestinal lumen, containing a wide variety of microbes, and the different layers of the intestinal wall, which contain numerous potent effector cells of the immune system. Intriguingly, specialized T lymphocyte subpopulations are regularly found within the intestinal epithelium. Under physiological conditions, approximately 10% of the cells of the epithelial layer are T cells, hence, termed intraepithelial lymphocytes. Considering the vast surface area covered by the intestinal epithelium (in humans approx. 300 m2), the total number of intraepithelial lymphocytes is impressive and easily exceeds the total number of T cells found in the spleen. 

In their phenotypic composition these intraepithelial lymphocytes differ dramatically from the T cells found in the peripheral blood and in lymphoid organs. In particular, besides the “conventional” CD8αβ TCRαβ T cells “unconventional” T cell subsets like TCRgd T cells, and CD8αα TCRαβ T cells (which also comprise autoreactive T cells) are present in the intestinal epithelium. Although in mice “conventional” CD8αβ TCRαβ IEL and “unconventionable” CD8αα TCRαβ IEL are present in comparable numbers in the small intestine, our knowledge on their function is still highly limited. This partial ignorance may be attributed in part to their poor survival in vitro upon ex vivo isolation.

Hence, in the present project we propose to: (i) establish an optimized in vitro culture system for intestinal CD8αα TCRαβ T cells; (ii) compare the functional properties and activities of autoreactive, vs. non-autoreactive CD8αα TCRαβ IEL; (iii) define the impact of a colonization of the gut lumen with a defined microbial flora on the functional differentiation of intestinal CD8αα TCRαβ IEL, and (iv), define the functional changes induced during an intestinal inflammation in CD8αα TCRαβ IEL.

This set of experiments should allow to obtain relevant information on the potential plasticity and/or functional heterogeneity of this still enigmatic T cell subset, and reveal their potential involvement in regulating inflammatory reaction in the intestinal mucosa, but possibly also at extraintestinal sites.


Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis
Zysset Daniel Weber Benjamin Rihs Silvia Brasseit Jennifer Freigang Stefan Riether Carsten Ban (2016), TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis, in Nature Communications, 7, 13151.
IFN-γ Hinders Recovery from Mucosal Inflammation during Antibiotic Therapy for Salmonella Gut Infection
Dolowschiak Tamas, Mueller AA, Pisan LJ, Feigelman R, Felmy Boas, Sellin ME, Namineni S, Nguyen BD, Wotzka SY, Heikenwalder M, von Mering Christian, Mueller Christoph, Hardt Wolf-Dieter (2016), IFN-γ Hinders Recovery from Mucosal Inflammation during Antibiotic Therapy for Salmonella Gut Infection, in Cell Host Microbe, 20(2), 238-249.
CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis.
Brasseit J, Althaus-Steiner E, Faderl M, Dickgreber N, Saurer L, Genitsch V, Dolowschiak T, Li H, Finke D, Hardt W-D, McCoy K D, Macpherson A J, Corazza N, Noti M, Mueller C (2016), CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis., in Mucosal immunology, 9(3), 689-701.
Quality of Life in Swiss Paediatric Inflammatory Bowel Disease Patients: Do Patients and Their Parents Experience Disease in the Same Way?
Mueller Rebekka, Ziade Farah, Pittet Valérie, Fournier Nicolas, Ezri Jessica, Schoepfer Alain, Schibli Susanne, Spalinger Johannes, Braegger Christian, Nydegger Andreas, Swiss IBD Cohort Study (2016), Quality of Life in Swiss Paediatric Inflammatory Bowel Disease Patients: Do Patients and Their Parents Experience Disease in the Same Way?, in Journal of Crohn's & colitis, 10(3), 269-76.
Symptoms of Depression and Anxiety Are Independently Associated With Clinical Recurrence of Inflammatory Bowel Disease.
Mikocka-Walus Antonina, Pittet Valerie, Rossel Jean-Benoît, von Känel Roland, Swiss IBD Cohort Study Group (2016), Symptoms of Depression and Anxiety Are Independently Associated With Clinical Recurrence of Inflammatory Bowel Disease., in Clinical gastroenterology and hepatology : the official clinical practice journal of the American Ga, 14(6), 829-835.
Impact of the early use of immunomodulators or TNF antagonists on bowel damage and surgery in Crohn's disease.
Safroneeva E, Vavricka S R, Fournier N, Pittet V, Peyrin-Biroulet L, Straumann A, Rogler G, Schoepfer A M, Swiss IBD Cohort Study Group (2015), Impact of the early use of immunomodulators or TNF antagonists on bowel damage and surgery in Crohn's disease., in Alimentary pharmacology & therapeutics, 42(8), 977-89.
Keeping bugs in check: The mucus layer as a critical component in maintaining intestinal homeostasis.
Faderl Martin, Noti Mario, Corazza Nadia, Mueller Christoph (2015), Keeping bugs in check: The mucus layer as a critical component in maintaining intestinal homeostasis., in IUBMB life, 67(4), 275-85.
Systematic analysis of factors associated with progression and regression of ulcerative colitis in 918 patients.
Safroneeva E, Vavricka S, Fournier N, Seibold F, Mottet C, Nydegger A, Ezri J, Straumann A, Rogler G, Schoepfer A M, Swiss IBD Cohort Study Group (2015), Systematic analysis of factors associated with progression and regression of ulcerative colitis in 918 patients., in Alimentary pharmacology & therapeutics, 42(5), 540-8.
Analysis of TNF-antagonist switch over time and associated risk factors in the Swiss Inflammatory Bowel Disease Cohort.
Hiroz Philippe, Vavricka Stephan R, Fournier Nicolas, Safroneeva Ekaterina, Pittet Valérie, Rogler Gerhard, Schoepfer Alain M, Swiss Inflammatory Bowel Diseases Cohort Study Group (2014), Analysis of TNF-antagonist switch over time and associated risk factors in the Swiss Inflammatory Bowel Disease Cohort., in Scandinavian journal of gastroenterology, 49(10), 1207-18.
Appropriateness and long-term discontinuation rate of biological therapies in ulcerative colitis.
Maillard MH, Bortolotti M, Vader JP, Mottet C, Schoepfer A, Goners JJ, Burnand B, Froehlich F, Michetti P, Pittet V, Swiss IBD Cohort Study Group (2014), Appropriateness and long-term discontinuation rate of biological therapies in ulcerative colitis., in J Crohns Colitis, 8(8), 825-834.
Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.
Thelemann Christoph, Eren Remzi Onur, Coutaz Manuel, Brasseit Jennifer, Bouzourene Hanifa, Rosa Muriel, Duval Anais, Lavanchy Christine, Mack Vanessa, Mueller Christoph, Reith Walter, Acha-Orbea Hans (2014), Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis., in PloS one, 9(1), 86844-86844.
Prevalence and clinical importance of mesenteric venous thrombosis in the Swiss Inflammatory Bowel Disease Cohort.
Violi N Vietti, Vietti Violi Naïk, Schoepfer Alain M, Fournier Nicolas, Guiu Boris, Bize Pierre, Denys Alban, Swiss Inflammatory Bowel Disease Cohort Study Group (2014), Prevalence and clinical importance of mesenteric venous thrombosis in the Swiss Inflammatory Bowel Disease Cohort., in AJR. American journal of roentgenology, 203(1), 62-9.
Prevalence and clinical importance of mesenteric venous thrombosis in the Swiss Inflammatory Bowel Disease Cohort.
Vietti Violi Naïk, Schoepfer Alain M, Fournier Nicolas, Guiu Boris, Bize Pierre, Denys Alban, Swiss Inflammatory Bowel Disease Cohort Study Group (2014), Prevalence and clinical importance of mesenteric venous thrombosis in the Swiss Inflammatory Bowel Disease Cohort., in AJR. American journal of roentgenology, 203(1), 62-9.
Serum ficolin-2 correlates worse than fecal calprotectin and CRP with endoscopic Crohn's disease activity.
Schaffer Thomas, Schoepfer AM, Seibold Frank, Swiss IBD Cohort Study Group (2014), Serum ficolin-2 correlates worse than fecal calprotectin and CRP with endoscopic Crohn's disease activity., in J Crohns Colitis, 8(9), 1125-1132.
Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.
Bjerrum Jacob T, Nielsen Ole H, Riis Lene B, Pittet Valerie, Mueller Christoph, Rogler Gerhard, Olsen Jørgen (2014), Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia., in Inflammatory bowel diseases, 20(12), 2340-52.
TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance.
Weber Benjamin, Schuster Steffen, Zysset Daniel, Rihs Silvia, Dickgreber Nina, Schürch Christian, Riether Carsten, Siegrist Mark, Schneider Christoph, Pawelski Helga, Gurzeler Ursina, Ziltener Pascal, Genitsch Vera, Tacchini-Cottier Fabienne, Ochsenbein Adrian, Hofstetter Willy, Kopf Manfred, Kaufmann Thomas, Oxenius Annette, Reith Walter, Saurer Leslie, Mueller Christoph (2014), TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance., in PLoS pathogens, 10(1), 1003900-1003900.
Diagnostic delay in Crohn's disease is associated with a complicated disease course and increased operation rate.
Schoepfer Alain M, Dehlavi Mohamed-Ali, Fournier Nicolas, Safroneeva Ekaterina, Straumann Alex, Pittet Valérie, Peyrin-Biroulet Laurent, Michetti Pierre, Rogler Gerhard, Vavricka Stephan R, IBD Cohort Study Group (2013), Diagnostic delay in Crohn's disease is associated with a complicated disease course and increased operation rate., in The American journal of gastroenterology, 108(11), 1744-1753.
Pancreatic stone protein as a novel marker for neonatal sepsis.
Schlapbach Luregn J, Graf Rolf, Woerner Andreas, Fontana Matteo, Zimmermann-Baer Urs, Glauser David, Giannoni Eric, Roger Thierry, Müller Christoph, Nelle Mathias, Stocker Martin (2013), Pancreatic stone protein as a novel marker for neonatal sepsis., in Intensive care medicine, 39(4), 754-63.
Penetrating or stricturing diseases are the major determinants of time to first and repeat resection surgery in Crohn's disease.
Pittet Valerie, Rogler Gerhard, Michetti Pierre, Fournier Nicolas, Vader John-Paul, Schoepfer Alain, Mottet Christian, Burnand Bernard, Froehlich Florian, Swiss Inflammatory Bowel Disease Cohort Study Group (2013), Penetrating or stricturing diseases are the major determinants of time to first and repeat resection surgery in Crohn's disease., in Digestion, 87(3), 212-21.
Predictors for hospitalization and outpatient visits in patients with inflammatory bowel disease: results from the Swiss Inflammatory Bowel Disease Cohort Study.
Sulz Michael C, Siebert Uwe, Arvandi Marjan, Gothe Raffaella M, Wurm Johannes, von Känel Roland, Vavricka Stephan R, Meyenberger Christa, Sagmeister Markus, Swiss IBD Cohort Study Group (2013), Predictors for hospitalization and outpatient visits in patients with inflammatory bowel disease: results from the Swiss Inflammatory Bowel Disease Cohort Study., in European journal of gastroenterology & hepatology, 25(7), 790-7.
Predictors of temporary and permanent work disability in patients with inflammatory bowel disease: results of the swiss inflammatory bowel disease cohort study.
Siebert Uwe, Wurm Johannes, Gothe Raffaella Matteucci, Arvandi Marjan, Vavricka Stephan R., Von Känel Roland, Begré Stefan, Sulz Michael Christian, Meyenberger Christa M., Sagmeister Markus, Swiss IBD Cohort Study Group (2013), Predictors of temporary and permanent work disability in patients with inflammatory bowel disease: results of the swiss inflammatory bowel disease cohort study., in Inflammatory bowel diseases, 19(4), 847-855.
Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.
Scharl Michael, Mwinyi Jessica, Fischbeck Anne, Leucht Katharina, Eloranta Jyrki J, Arikkat Joba, Pesch Theresa, Kellermeier Silvia, Mair Alma, Kullak-Ublick Gerd A, Truninger Kaspar, Noreen Faiza, Regula Jaroslaw, Gaj Pawel, Pittet Valerie, Mueller Christoph, Hofmann Claudia, Fried Michael, McCole Declan F, Rogler Gerhard (2012), Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy., in Inflammatory bowel diseases, 18(5), 900-12.
Danger-associated molecular patterns and inflammatory bowel disease: is there a connection?
Mueller Christoph (2012), Danger-associated molecular patterns and inflammatory bowel disease: is there a connection?, in Digestive diseases (Basel, Switzerland), 30 Suppl 3, 40-6.
Elevated levels of serum-soluble triggering receptor expressed on myeloid cells-1 in patients with IBD do not correlate with intestinal TREM-1 mRNA expression and endoscopic disease activity.
Saurer Leslie, Rihs Silvia, Birrer Michèle, Saxer-Seculic Nikolina, Radsak Markus, Mueller Christoph, Swiss IBD Cohort Study (2012), Elevated levels of serum-soluble triggering receptor expressed on myeloid cells-1 in patients with IBD do not correlate with intestinal TREM-1 mRNA expression and endoscopic disease activity., in Journal of Crohn's & colitis, 6(9), 913-23.
Entzündung
Müller Christoph, Holländer Georg, Imhof Beat, Höfler Gerhard (2012), Entzündung, in Werner Böcker Helmut Denk Philipp Ulrich Heitz Holger Moch Gerald Höfler Hans Kreipe (ed.), 43-73.
Pathologische Immunreaktionen
Müller Christoph, Holländer Georg, Imhof Beat, Höfler Gerhard (2012), Pathologische Immunreaktionen, in Werner Böcker Helmut Denk Philipp Ulrich Heitz Holger Moch Gerald Höfler Hans Kreipe (ed.), 75-111.
Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease.
Eloranta Jyrki J, Wenger Christa, Mwinyi Jessica, Hiller Christian, Gubler Christoph, Vavricka Stephan R, Fried Michael, Kullak-Ublick Gerd A (2011), Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease., in Pharmacogenetics and genomics, 21(9), 559-64.
Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis.
Philippe David, Favre Laurent, Foata Francis, Adolfsson Oskar, Perruisseau-Carrier Genevieve, Vidal Karine, Reuteler Gloria, Dayer-Schneider Johanna, Mueller Christoph, Blum Stéphanie (2011), Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis., in World journal of gastroenterology, 17(4), 459-69.
CX3CR1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions.
Weber Benjamin, Saurer Leslie, Schenk Mirjam, Dickgreber Nina, Mueller Christoph (2011), CX3CR1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions., in European journal of immunology, 41(3), 773-9.
Epidemiology of inflammatory bowel disease: Is there a shift towards onset at a younger age?
Braegger Christian P, Ballabeni Pierluigi, Rogler Daniela, Vavricka Stephan R, Friedt Michael, Pittet Valérie (2011), Epidemiology of inflammatory bowel disease: Is there a shift towards onset at a younger age?, in Journal of pediatric gastroenterology and nutrition, 53(2), 141-4.
HVEM signalling promotes colitis.
Schaer Corinne, Hiltbrunner Stefanie, Ernst Bettina, Mueller Christoph, Kurrer Michael, Kopf Manfred, Harris Nicola L (2011), HVEM signalling promotes colitis., in PloS one, 6(4), 18495-18495.
IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo.
Mudter Jonas, Yu Jingling, Zufferey Christel, Brüstle Anne, Wirtz Stefan, Weigmann Benno, Hoffman Arthur, Schenk Mirjam, Galle Peter R, Lehr Hans A, Mueller Christoph, Lohoff Michael, Neurath Markus F (2011), IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo., in Inflammatory bowel diseases, 17(6), 1343-58.
Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.
Batsford Stephen, Duermueller Ursula, Seemayer Christian, Mueller Christoph, Hopfer Helmut, Mihatsch Michael (2011), Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease., in Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant, 26(4), 1413-6.

Collaboration

Group / person Country
Types of collaboration
Swiss IBD Cohort Study (SNF # 3347CO-108792) Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Annette Oxenius, ETH Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Wolf-Dieter Hardt, ETH Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Fabienne Tacchini-Cottier Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Andrew Macpherson, Universität Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Walter Reith, Geneva Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Daniela Finke, University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Markus Neurath, Universität Erlangen Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Kati Andreasson, Stanford University Medical Center (Neurology and Neurosciences) United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Jahrestagung der Deutsche Arbeitsgemeinschaft für chronisch-entzündliche Darmerkrankungen DACED Talk given at a conference Different layers of immune protection in the mouse intestine (Keynote address) 19.06.2015 Mainz, Germany Müller Christoph;


Self-organised

Title Date Place
Day of Research, Institute of Pathology, University of Bern (including Scientific Board Meeting) 21.01.2015 Bern, Canton of Bern, Switzerland, Switzerland
Day of Research, Institute of Pathology, University of Bern (including Scientific Board Meeting) 25.01.2014 Bern, Switzerland
Day of Research, Institute of Pathology, University of Bern (including Scientific Board Meeting) 30.01.2013 Bern, Switzerland
Day of Research, Institute of Pathology, University of Bern (including Scientific Board Meeting) 29.02.2012 Bern, Switzerland

Communication with the public

Communication Title Media Place Year
Media relations: print media, online media Neue Erkenntnisse zur Entstehung der Arterienverkalkung Medienmitteilung Universität Bern International German-speaking Switzerland 2016
Media relations: print media, online media Geräuschloses Begräbnis (wissenschaftliche Beratung) NZZ German-speaking Switzerland 2014

Awards

Title Year
Honorary Member, Swiss Society for Allergology and Immunology 2016

Associated projects

Number Title Start Funding scheme
145006 Apparatus for Colonoscopy and Endoscopic Manipulations in Mice 01.09.2013 R'EQUIP
134274 Schweizer IBD Kohorten Studie 01.04.2011 Cohort Studies Large
145016 Bringing flow cytometry a step further: expanding biomedical research capabilities with an imaging flow cytometer 01.10.2012 R'EQUIP
170084 Intestinal tissue resident memory T cells in inflammation and homeostasis 01.11.2016 Project funding (Div. I-III)
136286 Recovery of intestinal homeostasis after microbial or immunological challenge 01.12.2011 Sinergia
170084 Intestinal tissue resident memory T cells in inflammation and homeostasis 01.11.2016 Project funding (Div. I-III)
122560 Immune reactions in the intestinal mucosa: pathophysiology of intraepithelial lymphocytes 01.10.2008 Project funding (Div. I-III)

Abstract

Summary BackgroundIntestinal CD8aa TCRaß T cells represent a major cell subset in the small and large intestine of mice. Despite their abundance in the intestinal epithelium, our knowledge on their antigen specificity, and the functions exerted in vivo are still limited. This may in part be ascribed to the poor survival of ex vivo isolated CD8aa TCRaß T cells in vitro. In their development they are distinct from conventional, MHC class I and class II restricted CD8aß, and CD4 TCRaß T cells. Intriguingly, CD8aa TCRaß T cells with an autoreactive TCRaß are not centrally eliminated during their intrathymic differentiation and persist in the intestinal epithelium. In contrast to the intestinal CD8aß TCRaß intraepithelial lymphocytes (IEL) CD8aa TCRaß IEL do not recirculate under homeostatic conditions and are bona fide resident T cells in the intestinal epithelium as evidenced by parabiosis experiments. Our preliminary data demonstrate an accumulation of CD8aa TCRaß T cells during a potent inflammation in the intestinal mucosa not only in the affected colonic mucosa, but also at extraintestinal sites (e.g. mesenteric and inguinal lymph nodes, spleen, blood). This dramatically altered biological behavior of the CD8aa TCRaß IEL is associated with a strong proliferation and a marked change in the cell surface expression pattern of chemotactic receptors.Working hypothesisCD8aa TCRaß T cells are functionally a heterogeneous population of T cells. Factors that influence their functional activities are the presence of self-antigens for autoreactive CD8aa TCRaß T cells; the presence of a (mostly) commensal microbiota (and their products); and potent inflammatory stimuli in the intestinal mucosa.Specific aims•Establishment of an optimized in vitro culture system for intestinal CD8aa TCRaß T cells.•Comparative analysis of the functional properties and activities of autoreactive, vs. non-autoreactive CD8aa TCRaß IEL.•Defining the impact of colonization of the gut lumen with a defined microbial flora (altered Schaedler flora, ASF) on the functional differentiation of intestinal CD8aa TCRaß IEL; assess TCRaß dependent, vs. TCRaß independent events.•Assessment of the functional and biological changes induced during intestinal inflammation in CD8aa TCRaß IEL, including the relative contribution of distinct chemotactic pathways to their altered migration during intestinal inflammation and an analysis of the molecular pathways that regulate functional, migratory and proliferative capacities in CD8aa TCRaß T cells.Experimental design•To determine the functional properties of bona-fide autoreactive vs. non-autoreactive CD8aa TCRaß IEL, the LCMV-gp33 transgenic mouse (H8 mouse), and the gp33 specific TCRaß transgenic mouse (318) on a RAG-/- background will be used for analysis. Monoclonal, autoreactive CD8aa TCRaß IEL will be obtained from double transgenic 318xH8 RAG-/- mice. The 318 RAG-/- mouse line, which lacks expression of the cognate antigen, will be used as donors of non-autoreactive CD8aa TCRaß IEL (upon FACS sorting), and intestinal CD8aa TCRaß IEL, will be isolated from H8 mice as a polyclonal population of autoreactive T cells based on gp33-tetramer staining. Methods for analysis of the different CD8aa TCRaß T cell populations include: global gene expression profiling (Affymetrix Mouse Exon 1.0 ST Arrays), FACS stainings for activation, differentiation and proliferation markers (panel of primary reagents will be adjusted also based on the results of mRNA profiling), qRT-PCR for selected cell subsets (e.g. from distinct anatomical locations), and in vitro cultures (e.g. restimulation for cytokine secretion, functional assays).•Germ-free mice (B6, Bim deficient mice) will be colonized with an altered Schaedler flora to determine the early functional and biological changes induced in the CD8aa TCRaß IEL through intestinal colonization of the gut lumen; CD8aa TCRaß IEL will be analyzed between 0 and 20 days post colonization. •To define the functional changes CD8aa TCRaß IEL by an intestinal inflammation, both, the chronic dextran sodium sulfate (DSS) model of colitis induction, and the CD4 T cell transfer model of colitis in the CD8aa TCRaß containing 318xH8 RAG-/- mice will be used; the relevance of the different chemotactic pathways for the migration of IEL subsets will be assessed in these colitis models using specific receptor deficient mice (e.g. CXCR3-/-; BLT1/Ltb4R-/-), or administration of an S1PR1 agonist.Expected value of the projectThe use of well defined experimental systems, particularly including gnotobiotic animals with a well defined microbiota, to address the specific aims in combination with in vitro cultures of (fractionated) CD8aa TCRaß cell subsets should allow to obtain relevant information on the potential plasticity and/or functional heterogeneity of this still enigmatic T cell subset, and reveal their potential involvement in regulating inflammatory reaction in the intestinal mucosa, but possibly also at extraintestinal sites. The functional characterization of CD8aa TCRaß IEL will be critical to assess their impact also on the functions of the neighboring IEC, a central issue, which so far has not been fully addressed.
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