Innate lymphoid cells (ILC) in immunity. Marco Colonna Washington University in St Louis

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1 Innate lymphoid cells (ILC) in immunity Marco Colonna Washington University in St Louis

2 Innate Lymphoid Cells (ILC) before 2009: Natural killer cell (NK) Inhibitory Receptor CD43 CD27 CD11b CD11c lo Activating Receptor NK1.1 + NKp46 CD94/NKG2x Ly49 Lymphoid tissue inducer cell (LTi) IFN-γ TNF-α GM-CSF CCL4 Perforin Granyzmes CD127 Id2 Rorγt CD4 α4β7 c-kit RET LTα 1 β 2 CXCL13 CCL21 CCL19 VCAM1 ICAM1 MADCAM1 IL-7 Cytotoxicity Lymphoid organogenesis

3 Innate Lymphoid Cells (ILC) after 2009 IL-15 IL-12 IL-18 NK ILC1 IFN-γ Intracellular pathogens autoimmunity IL-23 IL-1β ILC3 LTi IL-22 IL-17 (IFN-γ) Lymphoid organogenesis Extracellular bacteria fungi autoimmunity IL-25 IL-33 TSLP ILC2 IL-5 IL-9 IL-13 Amphiregulin Helminth Allergies Asthma

4 Functional modules of CD4 Th cells Inducing cytokines TF Effector cytokines Functional properties IFNγ + IL-12 T H 1 Tbet/ STAT4 IL-2, IFN-γ Phagocytic Intracellular pathogens Autoimmunity Naïve CD4 T cell IL-4 TGF-β IL-6 IL-1, IL-23 TGF-β T H 2 GATA3/ STAT6 T H 17 RORγT T Reg FOXP3 IL-4, IL-5 IL-13, Amphiregulin IL-17a/f, IL-22, IL-21 IL-10,TGFβ, IL-35 Mucosal Barrier Acute Inflammatory Regulatory Helminths Allergy and asthma Extracellular bacteria, fungi Autoimmunity Immune tolerance Lymphocyte homeostasis Regulation of immune responses

5 Transcription factors and cytokines in ILC development αlp Cytolytic Helper NKp46 + ILC3

6 ILCs are tissue resident cells s Skin +fat ILC1 ILC1 cnk cell cnk cell ILC1 ILC1 ILC2 ILC2 ILC3 ILC3 cnk cell cnk cell CD4+ LTi CD4+ LTi like CD4- LTi CD4- LTi like ex- Rorgt ILC3 ex- Rorgt ILC3 NKp46 ILC3 NKp46 ILC3 ILC3 ILC3 LTi LTi like uterine NK uterine NK ILC2 ILC2 ILC1 ILC1 cnk cell cnk cell ILC3 ILC3 Rare: IEL ILC1 IEL ILC1 Small intestine Colon Salivary Glands Liver Lung trachea bronchi Brain Spleen Bladder urethra Kidney Heart Pancreas SG NK SG NK ILC2 ILC2 Tonsils ILC1 ILC1 cnk cell cnk cell ILC3 ILC3 Heart

7 Similarity of functional modules in innate and adaptive immunity T H 1 CD8/4T RM T H 2 T H 17 NK ILC1 ILC2 ILC3 IFN-γ IFN-γ IL-5 IL-13 IL-22 IL-17

8 NK cells have multiple receptors m157 MCMV Ly49H DNA damage dsdna breaks RAE MULT1 ULBP NKG2D MIC Target ICAM B7H6 NKp46 NKp30 NKp44 CD48 EBV 2B4 CRACC NTB-A DAP12 DAP10 integrins CD3ζ DAP12 SAP EAT-2 NK cell

9 NKp44 is expressed in the human tonsil Tonsil CD3 lymphocytes CD56 NKp44

10 CD56+NKp44+ cells are found in human oral and gastrointestinal mucosae Surface mucosa Lamina propria Crypt mucosa Peyer s patches NKp44 Cytokeratin-5 appendix

11 Similarity between human NKp44+ cells and TH17/TH22 Type IL-17/22 CD4 T cells IL-23R CCR6 TCR IL-1R NKp44+ cells Innate lymphoid cells type 3 (ILC3) IL-23R NKp44 CCR6 IL-1R STAT3 activation TH17 TH22 RORγt NF-κB adjuvant effect STAT3 activation NKp44 + RORγt NF-κB adjuvant effect IL-17 and/or IL-22 IL-22 Cella, Fuchs et al. Nature. 2009;457:722

12 IL-22 triggers epithelial cell secretion of antimicrobial peptides and chemokines Sonnenberg GF, Nat Immunol 2011 IL-22 protects from bacterial infections but excessive secretion can cause inflammation (psoriasis)

13 Hypothesis: ILC3 protect the intestinal mucosa from bacterial infections Antimicrobial peptides CXCL1 CXCL1 Antimicrobial peptides Antimicrobial peptides CXCL1 CXCL1 IL-22 IL-22 IL-22 IL-22 IL-22 CCR6 CCR6 IL-26 APC JAML NKp44 CCR6 CD96 GPA33 IL-23/IL-1 NKp44+ cells (ILC3) Cella, Fuchs et al. Nature. 2009;457:722

14 Cooperation between ILC3 and T cells Antimicrobial peptides IL-22 IL-22 IL-17 IL-22 ILC3 Rorγt Ahr Th17 Rorγt Ahr Ahr T-bet Th22 IL-23/IL-1 TGF-β IL-23 IL-6 Th0 IL-6 MΦ DC infection DC d0 Innate immune response d7 Adaptive immune response

15 Mouse models for understanding the function of ILC3 Genetic models: Rag-/- x γc-/- Antibody depletion in RAG-/- mice anti-nk1.1 in Rag-/- mice anti-cd90 (anti-thy1) in Rag-/- mice Use of antibodies in Rag-/- mice or Rag-/- x γc-/- will deplete the cell population in question but will also eliminate other populations as well Need of lineage-specific deletion mutants

16 Which transcription factors are required for ILC3 development? Jacob Lee

17 NKp44+ cells express the transcription factor AHR Retinoic acid-related Orphan Receptors Aryl Hydrocarbon Receptor

18 Aryl Hydrocarbon Receptor (AHR): a sensor of polycyclic aromatic hydrocarbons

19 AHR ligands include nutritional components Acidic ph

20 Endogenous AHR ligands Tryptophan metabolism

21 Can we target AHR to block the development of ILC3? Analysis of conditional Ahr-/- mice Chris Bradfield

22 ILC3 subsets in mouse small intestine Human IL-23 IL-1β NKp44+ RORγT NKp46 + Rorγt+ Mouse IL-22 IL-22 Extracellular bacteria fungi autoimmunity Lti-like Rorγt+ Lymphoid organogenesis

23 ILC3 subsets in mouse small intestine Human IL-23 IL-1β NKp44+ RORγT NKp46 + Rorγt+ Mouse IL-22 IL-22 Extracellular bacteria fungi autoimmunity Lti-like Rorγt+ Lymphoid organogenesis

24 Conditional Ahr-deficient mice Ahr fl/fl x Roγt-cre

25 AHR is required for ILC3 development αlp Notch NKp46 + ILC3 Ahr

26 ILC3-deficient mice Mice AHR f/f x RORγt-Cre AHR f/f x RORγt-Cre x TCRβδ-/- Affected cells ILC3 ILC3, T cells RORγt f/f x NKp46-Cre NKp46 + ILC3 RORγt f/f x NKp46-cre x TCRβδ-/- NKp46 + ILC3, T cells RORγt f/f x NKp46-cre x Rag1-/- NKp46 + ILC3, T cells, B cells

27 Are ILC3 important for intestinal homeostasis?

28 Intestinal lymphoid tissues Peyer s Patches Develop pre-natally Eberl, Nat Rev Immunol 2005 CD3 B220

29 Intestinal lymphoid tissues ILF Develop post-natally CP Eberl, Nat Rev Immunol 2005 cryptopatches CD90+(Thy1+) c-kit+ Lti-ILC Isolated lymphoid follicles B220+/CD11c+

30 ILC3-deficient mice lack cryptopatches and isolated lymphoid follicles WT ILC3-deficient CD90+ (Thy1+) clusters identifying CP are absent in Ahr-deficient mice Prenatal lymphoid tissues (Peyer s Patches) are conserved CD90+ (Thy1+) c-kit+ clusters identifying CP are absent in Ahr-deficient mice CD11c+B220+ clusters identifying ILFs are absent in Ahr-deficient mice Rodney Newberry Keely McDonald

31 Are ILC3 important in host response to pathogenic bacteria? Christina Song

32 Citrobacter rodentium infection C. rodentium is a natural mouse pathogen related to enteropathogenic E. coli The infection is restricted to the lumen of the intestine In B6 mice, C. rodentium infection initiates on the surface of caecal patches, peaks at d7-14 pi and is cleared by d21-28 pi. Mundy R et al. Cell Microbiol. 2005;7:1697

33 Host defense against C. rodentium infection depends on IL-22 Zheng Y, et al. Nat Med. 2008;14:282.

34 C. rodentium infection in the absence of ILC3 and T cells AHR f/f x RORγt-cre x TCRβ/δ ko (no ILC3, no T cells) vs AHR f/f x TCRβ/δ-/- (no T cells) no T cells no ILC3, no T cells

35 C. rodentium infection in mice lacking ILC3 only AHR f/f x RORγt-cre (no ILC3) vs AHR f/f ILC3 protect the gastrointestinal tract from bacterial infections Partial redundancy with T cells ctr no ILC3

36 Do ILC3 contribute to IBD? IL-23R STAT3

37 ILC3 worsen anti-cd40-induced colitis α-cd40 0 AHR f/f x RORγt-cre x TCRβ/δ ko (no ILC3, no T cells) vs AHR f/f x TCRβ/δ-/- (no T cells) Body weight after anti-cd40 5-7d

38 Colon length 7 days after anti-cd40 Naive Anti-CD40 (d7) T cell Ctrl (Ahrf/f x TCRbd-/-) T cell ILC3 RORγt-cre x Ahrf/f x TCRbd-/- T cell T cell T cell T cell ILC3 NKp46 + ILC3 RORγt-cre x Ahrf/f x TCRbd-/- RORγtf/f x NKp46-cre x TCRbd-/- Ctrl (Ahrf/f x TCRbd-/-) Cre Ctrl (RORγt-cre+ x TCRbd-/-)

39 ILC3: two-edged sword Induce the development of ILF but have no obvious role in containment of commensal microbiota Protect from C. rodentium infections together with T cells Promote inflammation in a model of IBD

40 Similarity of functional modules in innate and adaptive immunity T H 1 CD8T RM T H 2 T H 17 NK ILC1 ILC2 ILC3 IFN-γ IFN-γ IL-5 IL-13 IL-22 IL-17

41 Human tonsils harbor 2 major subsets of NKp44 + ILCs Produce IL-22 upon stimulation with IL-23 NKp44 + ILC3 FL2-H: NKp44.PE NKp Localized within tonsil surface and crypt epithelium Secrete IFN-γupon stimulation with IL-12 + IL-15 Limited cytolytic capacity FL1-H: CD103.FITC CD103 Gated on CD56+ Anja Fuchs

42 Intraepithelial CD103 + ILC1 are present in human gastrointestinal tract Gated on CD45+CD3- lymphocytes Human appendix

43 Increased CD103 + ILC1 frequencies in the ileum of individuals with Crohn s disease NKp44 + CD103 + in IEL Gated on CD45+CD3- lymphocytes Ratio NKp44+CD103+ / CD3+ within IEL CD * NC Human appendix

44 NK cells or distinct ILC subset? FL2-H: NKp44.PE NKp CD103 FL1-H: CD103.FITC

45 Unique integrin pattern (VLA1+, CD103/Beta7+) NKp44+CD103+ ILC3 NKp44-(conventional NK)

46 Unique features: TGF-beta imprinting CD103 NKp44+CD103+ ILC3 NKp44-(conventional NK)

47 Similarity with Tissue Resident Memory CD8 T cells (T RM ) Memory RECIRCULATE T EM Home to peripheral tissue Pathogen Naive Intraepithelial Effector NKp44+CD103+ are tissue-resident ILC1 T CM Home to secondary lymphoid organs T RM Retained in peripheral tissues

48 Salivary gland NK cells are very similar to intestinal intraepithelial ILC1 Victor Cortez

49 NK cells vs ILC1 Recirculating Tissue resident NK IEL ILC1 uterus Liver ILC1 BM, spleen, LN, silp Intestinal epithelium also SG also silp and skin VLA2 positive negative positive* negative VLA1 negative positive positive positive TRAIL negative positive positive positive CD127 negative dim negative positive* IL-15 Dependent Partially independent Dependent Dependent Tbet Dependent Dependent Dependent Dependent Eomes Dependent Independent Independent Independent Nfil3 Partially independent Dependent Independent Dependent

50 Heterogeneity of ILC αlp iel ILC1 lp ILC1 liver ILC1 spleen ILC1 ILCs are complex! CD4+ CD4- Ahr Notch Ahr NKp46 + ILC3 ILC1 (ex-rorgt)

51 Understanding ILC diversity Unique and shared transcriptional programs of mouse ILCs In collaboration with the Immgen Consortium Michelle Robinette

52 ILC classes and subsets NK ILC2 ILC1 ILC3

53 ILC subsets frequency ILC2 ILC1 ILC1 NK NK ILC3 ILC1 NK

54 Principal component analysis of NK cell and ILC diversity

55 ILC1 and NK cells are intermixed Hierarchical clustering of the top 10% differentially expressed genes

56 Heatmap of ILC2 unique transcripts ILC2 IL4 IL5 IL13 IL9 IL9R IL33R M2-like polarization Arginase 1 PPARγ Dgat2 Lpcat2

57 Heatmap of ILC3 (Lti-like) unique transcripts ILC3 Lti Neuropilin 1

58 NKp46+ILC3 share transcripts with Lti ILC3 but also ILC1 ILC2 Lti ILC3 ILC1 ILC3 Nkp46+

59 Signature Transcription Factors Broad: Id2, GATA3, Nfil3 Small intestine lamina propria ILC and NK share an intestinal gene signature Class-specific: Eomes, Tbet, GATA3, Rorgt signature for silp: Rora, Atf3, Nr4a1

60 ILC1 transcriptional profile is intermediate between NK cells and ILCs slsilpilc1 Core ILC signature

61 ILC core signature: CXCR6 CXCR6 deficiency has no impact on ILC frequencies but influences cytokine responses

62 Conclusions ILC2 express transcripts indicative of M2 polarization Neuropilin1 is defining marker of Lti ILC3 NKp46+ ILC3 may have functional plasticity and convert into ILC1 Intestinal ILC and NK share an intestinal gene signature probably reflecting environmental exposure There is a major functional overlap between ILC1 and NK cells A core signature for ILC distinct from NK: CXCR6, IL-7R, GLT of TCRγ, TMEM176a/b

63 Lab 2014 Aaron Rapaport Alex Barrow Michelle Robinette Christina Song Luisa Cervantes Barragan Marina Cella Renata Sesti Satoru Yoshita Susan Gilfillan Tom Hannan Yaming Wang Victor Cortez

64 Acknowledgements Jacob Lee (Merck) Rodney Newberry Keely McDonald Thadd Stappenbeck ImmGen Collaborators Katerine Rothamel Christophe Benoist Funding R01DE R21CA16719 University of Brescia William Vermi University Toronto Tak Mak Marseille Luminy Eric Vivier University of Wisconsin Chris Bradfield