Shaping immunity in healthy and diseased tissues

Shaping immunity in healthy and diseased tissuesJannie Borst and Paola Ricciardi-Castagnoli

About one hundred immunologists recently met in Capo Caccia, Sardinia, for the firstof three events that will comprise the EMBO Conference Series, organised by theENII, on Molecular and Cellular Mechanisms of Immune Regulation. The 2007Conference focused on how immunity is shaped in healthy and diseased tissues.

Introduction

The immune response is a very dynamicprocess with distinct spatial and tempor-al components. A good understanding ofthe function and activation of individualcomponents of the immune system hasbeen reached and most of the moleculesand cells that are key players of animmune response have been character-ized. What is missing, however, is the“user manual” that permits reassemblyof the components to predict function-ing of the immune system as a whole.Combining current and new technolo-gies, such as genetic manipulation andimaging, allows this to be addressed andwas a theme of the meeting.

Imaging technologies were high-lighted in the Keynote lecture given byUlrich von Andrian (Boston, USA), oneof the leaders in the application ofintravital microscopy to track labelledcells in the living organism. His lectureincluded recent work on antigen-specificB cell trafficking in the mouse LN afterinjection of fluorescent vesicular stoma-titis virus (VSV) indicating that the virusaccumulates in the subcapsular areas ofthe LN, in macrophages that apparentlypresent it to B cells. Using VSV-specificand non-specific B cells of differentfluorescent colors, it was suggested thatonce the B cells have collected theantigen via the BCR and internalizedit, they relocalize to the T cell area.

Salvatore Valitutti (Toulouse,France) highlighted the spatial determi-nants of the immune response at the

cellular level in his State of the Artlecture. He reviewed the serial trigger-ing model of T cell activation andfeatured the immunological synapse,emphasizing that the synapse is adynamic structure both in helper andin cytotoxic T cells. Notably for cytotoxicT cells, lytic and stimulatory synapses,which depend on the antigen concen-tration, have been discerned [17].

Stem cells and lymphoidorganogenesis

The interaction of hematopoietic cellswith stromal cells appeared as a recur-rent theme of various presentations.Elaine Dzierzak (Rotterdam, The Neth-erlands) focused on hematopoiesis dur-ing embryogenesis and emphasized thatthe parallel development of mesenchy-mal and haematopoietic lineages is ageneral theme throughout ontogeny.She discussed the earliest events inhematopoiesis, which occur in theplacenta, the yolk sac and the AGM(aorta-gonad-mesonephros) region ofthe embryo [1]. She showed that withinthe AGM region, BMP4 allows mesench-ymal cells to commit to a hematopoieticfate. Subsequently, Tsvee Lapidot (Re-hovot, Israel) showed that the interac-tion of hematopoietic stem cells (HSC)with stromal cells, such as osteoblasts, inadult bone marrow niches is a dynamicprocess and that osteoclasts are involvedin HSC mobilization [2]. Factors such asCXCL12, which are produced in in-flamed organs, can reach the bone

marrow and mediate HSC mobilization.Reina Mebius (Amsterdam, The Nether-lands) and Dimitris Kioussis (London,UK), discussed the development of LNand Peyer's patches (PP) respectively.These processes depend on crosstalkbetween hematopoietic and mesenchy-mal cells, in which lymphotoxin plays acritical role [3]. The earliest hemato-poietic lymphoid tissue inducer cells(LTi) in the developing LN seem toexpress TRANCE, which may lead toupregulation of lymphotoxin; however,for PP development, the RET tyrosinekinase receptor signaling pathway, de-scribed to be involved in the develop-ment of the enteric nervous system andnow also shown to be required for thedevelopment of PP, was proposed to beinvolved in the induction of the firstlymphotoxin expression, in a novelsubset of CD11c+ LTi [4]. In bothdeveloping lymphoid organs, lympho-toxin will lead to the maturation ofmesenchymal cells to VCAM-1 expres-sing lymphoid tissue organizer cells.These cells can attract, by the productionof chemokines, more hematopoietic cellsto the developing secondary lymphoidorgans. Sanjiv Luther (Epalinges, Swit-zerland) subsequently showed that it iswithin these secondary lymphoid organsin adult animals, that fibroblastic re-ticular stromal cells provide survivalsignals to na�ve T lymphocytes, in theform of interleukin-7.

Crosstalk between infectious agentsand the host

Infection by microorganisms came intoplay with a focus on how such pathogenscan modulate responsiveness of theinfected cell. Olivier Neyrolles (Paris,

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France) emphasized the need for abetter understanding of immune regula-tion during infection with Mycobacter-ium tuberculosis. M. tuberculosis enterDC and macrophages via the moleculeDC-SIGN and hide in phagosomes,which do not acidify due to manipula-tion by the bacterial genome. Interest-ingly, macrophages and DC controlbacterial growth in different ways. DavidHolden (London, UK) came back to thistheme with his studies on Salmonella.This bacterium also remains long-termin the endocytic system of the host cell.The bacterial SPI-2 type III secretionsystem translocates various effectormolecules across the vacuole membraneinto the host cytoplasm, which preventsthe anti-bacterial response by interfer-ing with a diverse range of host cellprocesses [5]. Catharina Svanborg(Lund, Sweden) focused on pathogenicversus commensal Escherichia coli strainsin the urinary tract. Invasion of epithe-lial cells by pathogenic bacteria requiresTLR-4 expression [6]. Interestingly, thepathogenic bacteria can make a TLRmimetic that impairs TLR signalling andthus fails to alert the immune system.

The inflammatory responses

The earliest consequence of infection isthe activation of innate immune cellsand the inflammatory response. GeorgeKollias (Vari, Greece) presented hisstudies on genetically modified micewhich constitutively express TNF-a.These mice develop arthritis and/orinflammatory bowel disease (IBD).Gene expression profiling of synovialfibroblasts from healthy and diseasedmice revealed that TNF-a modulates thebehaviour of these cells, in particular byaffecting actin cytoskeleton dynamics.He also proved by conditional reactiva-tion of TNFR-1 on fibroblasts, that thesecells are sufficient targets for diseaseinduction by TNF. Alberto Mantovani(Milan, Italy) elegantly reviewed innatehumoral immunity including the longpentraxins, which are induced by IL-1 inmyeloid and mesenchymal cells and actin opsonization [7]. He introduced anovel type of IL-1 decoy receptor calledTIR8 and mice lacking this receptordevelop IBD and have increased suscept-ibility to colon cancer, highlighting theconnection between chronic inflamma-

tion and cancer. Interestingly, TIR8 hasan unusual cytoplasmic tail and acts asan intracellular decoy for signallingcomponents with affinity for TIR do-mains. Erwin Wagner (Vienna, Austria)showed that the transcription factorJunB is an important negative regulatorof inflammatory responses [8] and thatgenetic deletion of Jun and JunB in theepidermis leads to embryonic death.This was in spite of apparently normalbarrier function of the skin and due touncontrolled TNF-a production by theskin as elegantly demonstrated by ge-netic means.

Leukocyte recruitment andtrafficking

In this session aspects of leukocyteextravasation from the blood, leukocytemovement within tissues and their exitinto the lymphatic vessels were high-lighted. Dietmar Vestweber (M�nster,Germany) reported that the ESAMmolecule on endothelial cells is impor-tant for neutrophil diapedesis, as shownin genetically deficient mice and evi-dence was presented that signalling viathis molecule actively opens intercellu-lar junctions. CD99 on endothelial cellsis required for extravasation of bothlymphocytes and neutrophils; however,a new CD99-related and strongly O-glycosylated protein (CD99L2) presenton most leukocytes and endothelial cellcontacts was shown to participate inneutrophil extravasation but not lym-phocyte transmigration [9]. Lydia Sor-okin (M�nster, Germany) showed howthe macrophage-derived gelatinases(MMP-2 and MMP-9) cleave dystrogly-can and thus facilitate leukocyte extra-vasation through the blood-brain barrier[10]. Ronen Alon (Rehovot, Israel)provided evidence for the exciting con-cept that force is a critical switch inchemokine signalling [11]. Under flowconditions, chemokine receptors acti-vate LFA-1 and this starts lymphocytediapedesis. In the absence of flow,however, lymphocytes migrate usingthe immobilized CCR7 ligand only anddo not require integrin activation.Ronen Alon proposed therefore thatCCR7 coupling to LFA-1 is shut offduring the random walk of lymphocytesin LN. Shear stress or antigen receptorsignalling would install this coupling.

Sirpa Jalkanen (Turku, Finland) out-lined that little is known about lympho-cyte trafficking in and out of thelymphatic system. She reported the firstin vivo evidence of the relevance of themacrophage mannose receptor and CLE-VER-1 in lymphocyte trafficking via theafferent lymphatic vessels into thedraining LN. Nikolaus Romani outlinedthe travel of Langerhans cells (LC) fromthe epidermis to LN by scanning EM. Heemphasized that LC have to transversethe basement membrane and strugglethrough collagen to reach their destina-tion. Jean Davoust (Paris, France)tracked the same cells in situ by virtueof GFP expression under the Langerinpromoter [12]; under steady state con-ditions, LC are sessile, only undergoingtethered motions but under stress con-ditions virtually all become motile.Furthermore, LCwere shown tobe proneto interact with dying keratinocytes andmay cross present antigens derived fromthe apoptotic cells to CD8+ T cells.

Tissue specific immune activationand immune tolerance

Two sides of the same coin – themechanisms of activation and silencingof cells of the adaptive immune systemin response to infection or other chal-lenges – were discussed in a variety ofsites (skin, intestine, airways and thefetal/maternal interface), each withtheir own type of lymphoid organisa-tion. William Agace (Lund, Sweden)focused on the role of intestinal DC inthe generation of gut tropic T cells. Heprovided data to suggest that gut hom-ing receptors (CCR9 and a4b7) areinduced on T cells after activation by asubset of small intestinal DC expressingthe aIEL integrin subunit, CD103.CD103+ intestinal DC, but not non-intestinal or CD103– intestinal DC,induced retinoic acid receptor signalingin responding T cells, which appearscritical for induction of gut homingreceptors. He further showed an im-portant role for antigen dose in DC-mediated induction of tissue homingreceptors. David Adams (London, UK)focused on hepatic lymphocyte homingmechanisms and the role of intestinalprimed T cell populations in extraintest-inal manifestations of IBD. He demon-strated that in primary sclerosing cho-

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langitis (PSC), a disease that developswhen bowel inflammation is quiescentor after bowel removal, there is aberrantexpression of CCL25 in the liver andrecruitment of a CCR9+ a4b7+ memoryT cell population. He also showed thathuman intestinal LN, but not PSC liverDC, could induce CCR9 and a4b7 onresponding T cells, indicating that thesecells had been primed in intestinal LN.Nikolaus Romani (Innsbruck, Austria)and Tracy Hussell (London, UK) focusedon compartmentalized immunity in theskin and lung respectively. NicolasRomani provided evidence that LC havean important role in vivo [13] e.g. afterepicutaneous immunization against tu-mors. In the absence of LC, as achievedby LC ablation in mice expressing achimeric Langerin-diphtheria toxin re-ceptor, protection was lost. It was noted,however, that the functions of LC in vivo,in particular as compared to dermal DC,require further clarification.

Tracy Hussell presented the situationin different compartments of the lungand showed data supporting the ideathat excessive inflammation during re-spiratory infection is caused by a lack ofimmune organisation and is the result ofactivation of T cells by non-professionalAPC that are incapable of deliveringsubsequent inhibitory signals. Thismissing negative regulation of immunitycould be restored by blocking late T cellco-stimulatory molecules such as OX40or ligating the inhibitory receptorCD200R on APC; engagement ofCD200R on alveolar macrophages wasshown to downregulate the productionof inflammatory cytokines. Bart Lam-brecht (Rotterdam, The Netherlands)highlighted the significance of lung-resident DC. He showed spectacularintravital microscopy of lung DC pro-truding their dendrites in betweenepithelial cells and apparently “fishing”for antigen in the bronchial lumen. Heprovided evidence that the choice be-tweenT-cell tolerance or immunity uponintratracheal immunization with proteinantigen depends on the balance betweenplasmacytoid (p) and myeloid (m)DC;mDC prime the T cells, but pDC preventthem from becoming cytokine produ-cing effector cells. Virus infections breakthe tolerogenic function of pDC.

Bernd Arnold (Heidelberg, Ger-many) reminded us that parenchymal

cells can induce peripheral T cell toler-ance during neonatal life when thetissue is accessible to naive T cells. Toexamine their role in the adult, trans-genic mice expressing MBP peptidecovalently bound to MHC class IIb chain in keratinocytes were utilised.Upon skin irritation, the keratinocytesexpressed MHC class II a chain andpresented the peptide at the cell surface,and CD4+ T cells accumulated locally.Interestingly, although systemic res-ponses and autoimmunity in the shapeof EAE could be demonstrated, noautoimmune skin disease developed,suggesting that the skin micromilieucan downmodulate local acute inflam-mation. Reinhold F�rster (Hannover,Germany) reported that a variety ofimmune cells crucially rely on CCR7-directed migration not only for theinduction of immunity, but also forperipheral tolerance [14]. Active,CCR7-dependent transport of innocuousenvironmental antigen by resident DCfrom the periphery towards the lung andgut-draining LN is essential for theinduction of peripheral tolerance.Furthermore, using 2-photon micro-scopy it was observed that CCR7 andits ligands CCL21 and CCL19 contributeto the steady state intranodal motility ofna�ve T cells.

Finally, John Trowsdale (Cambridge,UK), described projects on sets ofimmune system genes (MHC, KIR) thatexhibit extreme polymorphism. Eightcommon haplotypes of the MHC havebeen completely sequenced by a con-sortium led by Stephan Beck of theSanger Centre in Cambridge in order totrack disease associations. The analysissuggested that some blocks of sequencediverged over 20 millions years ago asthey varied so greatly between haplo-types. Similarly, the KIR MHC class Ireceptors exhibit marked polymorphismwhich is interesting because poly-morphic MHC class I and KIR proteinsengage each other but are encoded ondifferent chromosomes. It was proposedthat HLA class I/KIR interactions haveconsequences for a range of infectiousand autoimmune disorders. In addition,work in Ashley Moffett's laboratorysuggests that KIR interactions betweenthe mother's NK cells and fetal-encodedHLA-C molecules influences conditionssuch as pre-eclampsia in which the fetus

does not have a sufficient blood supplyfrom the mother. Taken together withthe work from groups such as Eric Long'sand Ofer Mandelboim's, it was proposedthat, rather than influencing fetal toler-ance, class I/KIR interactions may play arole in vascularization to allow anadequate blood supply to the placenta.If HLA-C/KIR interactions regulate vas-cularization, this suggests that MHC andKIR polymorphisms may be driven notonly by immunity to infection but also byreproductive success.

Tumour tolerance and immunity

This session covered important topicsrelated to the basic mechanisms oftumor recognition, preclinical modelsof immunotherapy, as well as the latestefforts to translate the findings intoclinical applications. Immunosurveil-lance of cancer was addressed first byOlivera Finn (Pittsburgh, USA). Sheupdated the participants on her workon two tumor antigens, MUC1 and cyclinB1, which belong to the category of self-molecules aberrantly expressed on tu-mor cells. Antibodies and T cells specificfor the aberrant forms of these mole-cules can be found in many cancerpatients and, in the case of MUC1, whichhas beenmore extensively studied, theseimmune responses are associated withbetter prognosis. Olivera Finn's team hasdiscovered that close to 20% of healthyindividuals have immune responsesagainst these molecules. The theme ofimmunosurveillance was further devel-oped by Cornelis Melief (Leiden, TheNetherlands) who showed that thepresence of a large number of CD8+ Tcells infiltrating early stage cervicalcancer was associated with a lack oftumor metastasis in the draining LN.This in turn was associated with betterclinical outcome. Patients showingstrong CD8+ T cell infiltration intotumors also had circulating HPV-specificT cells presumably generated during theinitial viral infection [15]. CornelisMelief also presented early analysis ofa clinical trial of the HPV peptide vaccinetested in women with premalignantintraepithelial cervical neoplasm. Thevaccine was able to boost HPV-specific Tcell immunity and in some women thepremalignant lesions completely disap-peared. Finally, Beno�t van den Eynde

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(Brussels, Belgium) addressed the abil-ity of tumors to defeat the immunesystem, describing his research onindoleamine 2,3-dioxygenase (IDO) thatsuppresses the function of T cells. Thepresence of IDO was initially detected inmelanomas, but since then it has beenidentified in many other tumors andadding inhibitors of this enzyme tovaccines or other cancer immunothera-pies may improve their outcome. An-other attractive target of immunother-apy in the tumor microenvironment isthe tumor vasculature and G�nterH�mmerling (Heidelberg, Germany)shed new light on the nature of thetumor neovasculature [16]. Using atransgenic mouse model and carcino-gen-induced tumors he showed thattumors attract endothelial precursorcells (EPC) from the bone marrowthrough secretion of CC chemokines.These precursors are incorporated intothe tumor vasculature, promoting fastergrowth. This autocrine regulation ofneovascularization is a late event incarcinogenesis and it clearly facilitates acontinuous supply of nutrients and anexpansion of tumor size. Inhibitors of CCchemokines or blockers of chemokinereceptors on EPC are another combina-tion cancer immunotherapy approach.

Conclusion

Seeing immune cells in flagrante delicto,interacting with their tissue environ-ment, often in real time, added greatexcitement to this meeting. It is clearthat we have just started to face thecomplexity of studying the immuneresponse in the context of the tissueswhere immune cells are trafficking;however, the technical requirementsare in place, as is the theoretical frame-work to ask the important questions.Such in vivo work will undoubtedlybring interesting surprises.

Acknowledgements: The authors wish tothank the following speakers for their inputinto this report Reina Mebius, William Agace,Bernd Arnold, Olivera Finn and Sirpa Jalk-anen. Jannie Borst is at The NetherlandsCancer Institute, Amsterdam. The presentaddress of Paola Ricciardi-Castagnoli is SIgNat A*STAR, Singaporee-mail: [email protected]

Correspondence: Paola Ricciardi-Castagnoli,University of Milano-Bicocca, Dept. of Bio-technology, Piazza della Scienza 2, Milano,ItalyFax: +390-264483552e-mail: [email protected]

Abbreviations: IBD: inflammatory boweldisease � PP: Peyer's patches � LC: Langerhanscells � VSV: vesicular stomatitis virus

Meeting URL:http://www.enii.org/index.php?pageId=60

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Shaping immunity in healthy and diseased tissues