Innate and Innate-like Lymphocytes
in Allergic Inflammation
Jaichat Mekaroonkamol, MD.
outline
• Introduction
• Innate-like T cells : iNKT
– What is iNKT cells
– iNKT in asthma
• Innate lymphoid cells : ILC2
–...
Innate vs Adaptive immunity
Innate and Innate-like lymphocytes
Innate Immune
Cells
Adaptive Immune
Cells
ILCILC
Innate-like TInnate-like T
Innate-like...
Humoral immune response
• T dependent responses
• T independent responses
Abbas. Cellular and Molecular immunology. Sevent...
B lymphocyte subsets
Abbas. Cellular and Molecular immunology. Seventh Edition
B-1 B cellsB-1 B cells
• Self-renewing and preferentially produced in the
fetus
• Spontaneously produce polyreactive Igs
•...
Marginal Zone B cells
• Found in the marginal zone of the spleen
– May also be found in the blood
• Respond quickly to ant...
Characteristics of
T, innate-like T and Innate lymphoid cells
Conventional T
cells
Innate-like T
cells
Innate lymphoid
cel...
NKT cells
Natural Killer T (NKT) Cells
Unique subset of T cells
Function as innate cells, rapidly recognizing pattern
of antigens an...
Emmanuel Tupin, Yuki Kinjo & Mitchell Kronenberg
Nature Reviews Microbiology 5, 405-417 (June 2007)
iNKT Antigen Recogniti...
•Potent and specific activator of
iNKT cells
•Isolated from marine sponge
α-Galactosylceramide (α-GalCer)
Kawano Science 1...
α-GalCer and analogs
GalCer closely related structures forα
lipopolysaccharide (LPS) and -proteobacteriaα
Natural ligands
-proteobacteriaα
•Rickettsiales, Borrelia burgdorferi: Lyme disease,
Mycobacterium
Patrick J. Brennan et al. NATURE REVIEWS IMMUNOLOGY, FEBRUARY 2013.
iNKT cells during
microbial infecti
on.
Dingding An et al. Cell 156, 123–13
.
J Allergy Clin Immunol 2013;131:1393-9
But….
• In some circumstances, NKT cells can be
activated without the presence of lipid Ag
• How could they be activated?
Activation of iNKT cells
Patrick J. Brennan et al. NATURE REVIEWS IMMUNOLOGY, FEBRUARY 2013.
IL-12,
IL-18
Interactions between iNKT cells and other
leukocytes.
Patrick J. Brennan et al. NATURE REVIEWS IMMUNOLOGY, FEBRUARY 2013.
Borg Nature 2007, Scott-Browne Nat Imm 2007, Bendelac Review 2007, Brennan Nat Rev Imm 2013
AsthmaTumors Infections Autoim...
iNKT cells in AsthmaiNKT cells in Asthma
Heterogeneity of asthmaHeterogeneity of asthma
1. Antigen-specific
Th2 cells
2. Eosinophil
Other pathways beyond Th2 cell and
eosinophil??
Other pathways beyond Th2 cell and
eosinophil??
1. Non-allergic asthma is ...
iNKT cells and asthma
Wild type
vsvs
Exacerbate
/Ameriorate
Protective
/detrimental
vsvs
Anti-CD1d
blocking Ab
iNKT defici...
Everett H. Meyer. Annu. Rev. Med. 2008. 59:281–92
P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009.
NKT cells Function in Asthma
ILC2
P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009.
Chronic lung disease
Cytotox...
Protein allergens
α-GalCer
Virus Air pollution
Adapted from Busse NEJM 2003
iNKT
Lipid Ag
Targeting NKT cells for asthma t...
iNKT cells in human asthma
P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009.
At least 13 studies
-number of iNKT...
Functional of iNKT cells in human
P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009.
• Functional in vivo studies...
outline
• Innate-like T cells : iNKT
– What is iNKT cells
– iNKT in asthma
• Innate lymphoid cells : ILC2
– What is ILC2
–...
Th2 cytokines orchestrate allergic inflammation
Kay AB., NEJM 2001, Vol. 344 (1), 30-37.
IL-4
IL-5
IL-9
IL-13IL-13 Mucous ...
Th2 cytokines orchestrate allergic inflammation
Kay AB., NEJM 2001, Vol. 344 (1), 30-37.
IL-4
IL-5
IL-9
IL-13IL-13 Mucous ...
Innate Lymphoid Cells (ILC) secrete Th cytokines
Jennifer A. Walker et al. NATURE REVIEWS, 2013.
Innate Lymphoid Cells (ILC) secrete Th cytokines
Jennifer A. Walker et al. NATURE REVIEWS, 2013.
IL-5, IL-9, IL-13,
small ...
Jennifer A. Walker et al. NATURE REVIEWS, 2013.
Mary Elizabeth Jones and Yuan Zhuang. V(D)J recombination. Landes Bioscience ,2009.
ID2: transcriptional repressor inhibitor of DNA binding 2
Nature Reviews Cancer 14, 77–91: 2014.
“Natural helper cells”
Moro Nature Jan 2010
The absence of NHC in ID2 deficient mice
suggests that ID2 is important for ty...
Hergen Spits et al. NATURE REVIEWS IMMUNOLOGY, January 2013
Jennifer A. Walker et al. NATURE REVIEWS, 2013.
Hergen Spits et al. NATURE REVIEWS IMMUNOLOGY, January 2013
Discovery of ILC2 cells
RAG-deficient miceRAG-deficient mice
The Mechanism of V(D)J Recombination
Abbas. Cellular and Molecular immunology. Seventh Edition
Adapted from: Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. (without permission)
Th2 response
Fallon et al. The Journal of Experimental Medicine: April 10, 2006.
Adapted from: Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. (without permission)
“Nuocytes”
Neill Nature April 2010 Saenz Nature April 2010
Mice lacking IL-25 or IL-33 receptors have
very few nuocytes an...
Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013.
Type 2 Innate Lymphoid Cells (ILC2)
Adapted from: Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. (without permission)
Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013.
ILC2 in human also produce IL-13 in response to IL-25, IL-33ILC2 in human also produce IL-13 in response to IL-25, IL-33
M...
Innate type 2 cells in type 2 immune responsesInnate type 2 cells in type 2 immune responses
Neill, Trends in Parasitology...
ILC2 IN (HETEROGENEITY OF) ASTHMA
•GWAS of asthma consistently implicate IL-33 and
ST2 (Moffatt MF, Nature, 2007. Moffatt ...
Chang YJ., Nature Immunology, 2011
Influenza induces asthma independent of T cells
But dependent on ILC2
Influenza induces asthma independent of T cells
But ...
AM produce IL-33, increase ILC2 that produce IL-13
essential for H3N1-induced AHR.
AM produce IL-33, increase ILC2 that pr...
Influenza induce lung ILC to help
repair and remodeling
Nature Immunology 12, 1025–1027 (2011)
Influenza induce lung ILC to help
repair and remodeling
Influenza
IL-13
Amphiregulin
AHR
Airway inflammation
Klien, Curr A...
Glycolipid induced asthma depends on IL-33/ST2, ILC2 and
iNKT cells (independent of adaptive immunity)
-lipids found in mi...
Lipid recognitionLipid recognition
+
Kim HY. JACI Jan 2012
NKT cells Function in Asthma
ILC2
P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009.
Halim T. Immunity March 2012
•Identified and detailed characterized lung natural
helper cells
•Use protease allergen (papa...
ILC2-/-ILC2-/-
ILC2 are required for papain induced airway inflammationILC2 are required for papain induced airway inflamm...
ILC2 in fungal induced asthma modelILC2 in fungal induced asthma model
Bartemes, JI, 2012
Alternaria induces IL-33 in BAL
...
LTD4 potentiate Alternaria induced
asthma through ILC2 cysLT1R
LTD4 potentiate Alternaria induced
asthma through ILC2 cysL...
•ILC2 express ALX/FPR2 (receptor for Lipoxin A4)
•Lipoxin A4 can suppress IL-13 production by ILC2 in
response to PGD2 +/-...
Stokes Peebles, Sci Transl Med, 2013
ILC2 in the lungs
M2
AM
NKT
Pneumocyte
type2
Hardman, EJI, 2013
Chang, Nat Imm, 2011
Gorski, PlosPathogen, 2013
Nussbaum, ...
Conclusion
• Mechanism of allergic inflammation beyond Th2
• Roles of ILC2 & iNKT cells
• Importance of innate and innate-...
Innate and innate like lymphocytes in allergic inflammation
Innate and innate like lymphocytes in allergic inflammation
Innate and innate like lymphocytes in allergic inflammation
Innate and innate like lymphocytes in allergic inflammation
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Innate and innate like lymphocytes in allergic inflammation

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Innate and innate like lymphocytes in allergic inflammation

Presented by Jaichat Mekaroonkamol, M.D.

June20, 2014

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  • Two signals are involved in the physiological activation of
    invariant natural killer T (iNKT) cells: a T cell receptor (TCR) signal provided by a lipid–CD1d complex; and a cytokine signal
    that depends on the constitutive expression of certain cytokine receptors by iNKT cells. The left panel shows activation
    by a strong foreign antigen, which is dominated by the TCR signal and has little dependence on antigen-presenting cell
    (APC)-derived cytokines that are generated in response to the stimulation of pattern-recognition receptors (PRRs). By
    contrast, the right panel shows cytokine-dominated iNKT cell activation. In this scenario, PRR-mediated activation of APCs
    leads to the generation of pro-inflammatory cytokines such as interleukin‑12 (IL‑12). For cytokine-mediated activation, a
    TCR signal is still required in most cases and can be provided by a low-affinity microbial lipid antigen or self lipid antigen.
    The relative contributions of TCR and cytokine signals to iNKT cell activation are likely to be context dependent
  • The presentation of self or foreign lipid antigens
    facilitates cognate interactions between invariant natural killer T (iNKT) cells and CD1d‑expressing antigen-presenting
    cells (APCs), including dendritic cells (DCs), macrophages, neutrophils and B cells. This leads to iNKT cell activation and
    reciprocal APC activation or modulation. The major iNKT cell-derived cytokines that modulate the functions of the iNKT
    cell cognate partner during immune activation are shown. Both iNKT cell-derived factors and APC-derived factors can
    activate other cell types — such as natural killer (NK) cells and MHC-restricted T cells — in a CD1d‑independent manner.
    For example, DCs activated by iNKT cells secrete interleukin‑12 (IL‑12) to transactivate NK cells, and they can also
    enhance the ensuing adaptive immune responses mediated by MHC-restricted T cells. CD40L, CD40 ligand; CXCL2,
    CXC-chemokine ligand 2; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFNγ, interferon‑γ; IL‑12R, IL‑12
    receptor; TCR, T cell receptor; TNF, tumour necrosis factor
  • So, NKT cells seem to be a common pathways in various form of asthma.
    This knowledge open up exciting new areas of investigation in asthma pathogenesis and new potential therapeutic targets for asthma!!
  • Invariant natural killer T cells (iNKT) cell function in airway
    hyperreactivity (AHR). iNKT cells secrete various cytokines, including
    Th2 cytokines, which have direct effects on hematopoietic cells, airway
    smooth muscle cells, and goblet cells. Alternatively, iNKT cells could
    regulate other cell types that are known to be involved in asthma
    pathogenesis, e.g., neutrophils and alveolar macrophages.
  • So, NKT cells seem to be a common pathways in various form of asthma.
    This knowledge open up exciting new areas of investigation in asthma pathogenesis and new potential therapeutic targets for asthma!!
  • High levels of inhibitor of DNA binding (ID) proteins are present in stem and progenitor cells, where they sequester ubiquitously expressed basic helix–loop–helix (bHLH) transcription factors (also known as E proteins) and ETS transcription factors and thereby inhibit the transcription of lineage-specific and cell cycle-inhibitory genes12, 18, 19, 20, 25, 27. ID2 has been shown to interact with the tumour-suppressor protein RB, but hyperphosphorylated, inactive RB is incapable of binding to ID2 (Refs 29,30,31,32). b | With the downregulation of ID proteins (indicated by the smaller size of the ID shape), E proteins and tissue-specific bHLH (TS bHLH) transcription factors heterodimerize and bind to E-box sites in the promoters of target genes. ETS transcription factors are also released from the inhibitory activity of ID proteins and activate transcription. Low levels of ID2 are controlled by active hypophosphorylated RB. The transcription of lineage-specific genes and cell cycle-inhibitory genes is activated with a resulting loss of stem cell properties and acquisition of differentiated features and cell cycle arrest38, 104. P, phosphate
  • Initiation and propagation of type 2 responses. Type 2 responses
    are initiated by allergens or helminths that disrupt the epithelial barriers
    and induce secretion of IL-25, IL-33 and TSLP. Those epithelium-derived
    cytokines activate ILC2 cells, which directly secrete type 2 cytokines,
    and DCs, which induce TH2 responses. The secretion of type 2 cytokines
    by ILC2 cells feeds back on the epithelium to induce mucus secretion
    by goblet cells (IL-13) and tissue repair (amphiregulin (Areg)). Secretion
    of IL-9 and IL-5 by ILC2 cells leads to the recruitment and activation of
    mast cells and eosinophils. The activation of T cells in lymphoid organs
    further amplifies the secretion of type 2 cytokines, and the production of
    IL-4 by T cells in lymphoid organs leads to the production of IgE by
    B cells. Together, the responses triggered by secretion of type 2 cytokines
    from both ILC2 and TH2 cells orchestrate allergic inflammation, helminth
    expulsion and tissue repair
  • Initiation and propagation of type 2 responses. Type 2 responses
    are initiated by allergens or helminths that disrupt the epithelial barriers
    and induce secretion of IL-25, IL-33 and TSLP. Those epithelium-derived
    cytokines activate ILC2 cells, which directly secrete type 2 cytokines,
    and DCs, which induce TH2 responses. The secretion of type 2 cytokines
    by ILC2 cells feeds back on the epithelium to induce mucus secretion
    by goblet cells (IL-13) and tissue repair (amphiregulin (Areg)). Secretion
    of IL-9 and IL-5 by ILC2 cells leads to the recruitment and activation of
    mast cells and eosinophils. The activation of T cells in lymphoid organs
    further amplifies the secretion of type 2 cytokines, and the production of
    IL-4 by T cells in lymphoid organs leads to the production of IgE by
    B cells. Together, the responses triggered by secretion of type 2 cytokines
    from both ILC2 and TH2 cells orchestrate allergic inflammation, helminth
    expulsion and tissue repair
  • Initiation and propagation of type 2 responses. Type 2 responses
    are initiated by allergens or helminths that disrupt the epithelial barriers
    and induce secretion of IL-25, IL-33 and TSLP. Those epithelium-derived
    cytokines activate ILC2 cells, which directly secrete type 2 cytokines,
    and DCs, which induce TH2 responses. The secretion of type 2 cytokines
    by ILC2 cells feeds back on the epithelium to induce mucus secretion
    by goblet cells (IL-13) and tissue repair (amphiregulin (Areg)). Secretion
    of IL-9 and IL-5 by ILC2 cells leads to the recruitment and activation of
    mast cells and eosinophils. The activation of T cells in lymphoid organs
    further amplifies the secretion of type 2 cytokines, and the production of
    IL-4 by T cells in lymphoid organs leads to the production of IgE by
    B cells. Together, the responses triggered by secretion of type 2 cytokines
    from both ILC2 and TH2 cells orchestrate allergic inflammation, helminth
    expulsion and tissue repair
  • Initiation and propagation of type 2 responses. Type 2 responses
    are initiated by allergens or helminths that disrupt the epithelial barriers
    and induce secretion of IL-25, IL-33 and TSLP. Those epithelium-derived
    cytokines activate ILC2 cells, which directly secrete type 2 cytokines,
    and DCs, which induce TH2 responses. The secretion of type 2 cytokines
    by ILC2 cells feeds back on the epithelium to induce mucus secretion
    by goblet cells (IL-13) and tissue repair (amphiregulin (Areg)). Secretion
    of IL-9 and IL-5 by ILC2 cells leads to the recruitment and activation of
    mast cells and eosinophils. The activation of T cells in lymphoid organs
    further amplifies the secretion of type 2 cytokines, and the production of
    IL-4 by T cells in lymphoid organs leads to the production of IgE by
    B cells. Together, the responses triggered by secretion of type 2 cytokines
    from both ILC2 and TH2 cells orchestrate allergic inflammation, helminth
    expulsion and tissue repair
  • a) Change in lung resistance (RL) in 8-week-old BALB/c mice (n = 7–9 per group; anesthetized, tracheotomized, intubated and mechanically ventilated) challenged with methacholine nebulized into the airways 5 d after infection with H3N1 or allantoic fluid (mock infection control (mock)). *P < 0.01 and **P < 0.001, compared with mock infection (two-way analysis of variance (ANOVA)). (b) Macrophages (MΦ), neutrophils (Neu), eosinophils (Eos) and lymphocytes (Lym) in BAL fluid 5 d after treatment as in a. ND, not detectable. *P < 0.001, compared with mock infection (Student's two-tailed t-test). (c) Lung sections obtained from mock- or H3N1-infected BALB/c or C57BL/6 mice, stained with hematoxylin and eosin. Scale bars, 200 μm. (d) Change in lung resistance in 8-week-old C57BL/6 mice (n = 4 per group), treated and assessed as in a. *P ≤ 0.05 and **P ≤ 0.001. (e) Cells in BAL fluid 5 d after treatment as in d. *P < 0.001 (Student's two-tailed t-test). (f,g) AHR in 8-week-old wild-type (WT) or Rag2−/− mice (n = 6–7 per group) 5 d after infection with H3N1 or allantoic fluid, assessed as change in lung resistance (f) or cells in BAL fluid (g), as in a,b. *P ≤ 0.01 and **P ≤ 0.001 (two-way ANOVA (f) or Student's two-tailed t-test (g)). Data are representative of three independent experiments (mean and s.e.m.).
  • (a,b) Change in lung resistance (a) and cells in BAL fluid (b) in 8-week-old Rag2−/− mice (n = 4 per group) left undepleted (mock and H3N1) or depleted of CD90.2+ (Thy-1.2+) cells by three injections of mAb to CD90.2 (30-H12; 0.5 mg per mouse) on days −3, 0 and +3 (H3N1 + mAb to CD90.2), and infected with H3N1 or mock infected on day 0, analyzed 5 d after infection (as in Fig. 1a,b). *P < 0.05 and **P < 0.001, compared with H3N1 infection (two-way ANOVA (a) or Student's two-tailed t-test (b)). (c,d) Change in lung resistance (c) and cells in BAL fluid (d) in Il13−/− recipients (n = 4 per group) given purified natural helper cells (Lin−ST2+ subsets) from Il13+/+ (Rag2−/−) donors or Il13−/− donors treated intranasally with IL-33 (1 μg) 5 d before adoptive transfer of cells (1× 105 cells/mouse) by intratracheal injection, followed by mock infection or infection of recipients with H3N1 and analysis 5 d after infection (as in Fig. 1a,b). *P ≤ 0.001 (two-way ANOVA (c) or Student's two-tailed t-test (d)). Data are representative of two (a,b) or three (c,d) independent experiments (mean and s.e.m.).
  • After injury with an infectious agent such as influenza A, airway epithelial cells release IL-25 and IL-33; these induce ILC2 (lineage-negative (Lin−)) cells to produce amphiregulin, which serves to initiate epithelial-cell proliferation and repair through the activation of its receptor, EGFR. Simultaneously, ILC2 cells produce the prototypical type 2 cytokines (IL-5 and IL-13) that drive the symptoms of allergic disease, including goblet-cell metaplasia and airway hyper-responsiveness. The increase in IL-25 and IL-33 can perpetuate the allergic response by inducing further production of type 2 cytokines by basophils (Baso) and eosinophils (Eos) or by initiating the population expansion of conventional TH2 cells through the activation of dendritic cells (DC) during encounters with allergens. IFN, interferon; T1-ST2, mouse IL-33R subunit
  • Upon activation by glycolipid antigens, NKT cells induce macrophages, DCs and Type II
    pneumocytes to produce IL-33, which in turn activates natural helper cells and NKT cells to
    produce IL-13, resulting in the development of AHR. IL-33 can also activate mast cells,
    eosinophils and basophils
  • Invariant natural killer T cells (iNKT) cell function in airway
    hyperreactivity (AHR). iNKT cells secrete various cytokines, including
    Th2 cytokines, which have direct effects on hematopoietic cells, airway
    smooth muscle cells, and goblet cells. Alternatively, iNKT cells could
    regulate other cell types that are known to be involved in asthma
    pathogenesis, e.g., neutrophils and alveolar macrophages.
  • FIG 3. LTD4 induces ILC2 TH2 cytokine production through CysLT1R. A, Lung ILC2s were expanded in vivo after 3 Alternaria species challenges, followed by FACS and resting for 40 hours before in vitro studies. Presort (top) and postsort (bottom) ILC2 percentages are shown. B and C, IL-5 and IL-13 (Fig 3, B) and IL-4 (Fig 3, C) levels determined by using ELISA from supernatants of purified ILC2s stimulated with IL-33 or LTD4 (1026 and 1028 mol/L) for 6 hours with and without 1 mmol/L montelukast before treatment for 2 hours. D, ILC2 Fluo-4 intensity over time in seconds after 1026 mol/L LTD4 was added at 60 seconds without montelukast (left) and after pretreatment with montelukast (right). Data in Fig 3, A and D, are representative of 2 independent experiments, and data in Fig 3, B and C, are combined from triplicate wells of each condition of 2 independent experiments. MK, Montelukast. **P < .005 compared with media and #P < .01 compared with the same dose of LTD4 alone, Mann-Whitney test.
  • Figure 6. Pulmonary ILC2s are major Th2 cytokine producers in OVA-induced asthma. (A) Flow-cytometric analysis of intracellular cytokine content in OVA-challenged mice is shown. Total lung cell suspensions were stimulated with PMA/ionomycin for 4 h. ILC2s were gated as Lin− CD25+ CD127+ T1/ST2+ . Th cells were gated as T1/ST2+ or T1/ST2− CD3+ CD4+ lymphocytes. Values indicate the proportions cells ± SEM positive for the indicated cytokines. Plots are from representative samples of seven animals per group. (B) Contribution of lymphocyte populations indicated in panel A to production of IL-4 and IL-5, IL-13 in lungs, and BAL fluid, upon OVA treatment in vivo. Data are shown as mean + SEM of n = 7 animals. Experiment was performed once.
    Figure 5. Pulmonary ILC2s are major Th2 cytokine producers in HDM asthma. (A) Asthma protocols showing sensitization (S) and challenges (C) with house dust mite (HDM) in BALB/c mice, followed by analysis (A). Control mice were treated with PBS instead of HDM. (B) Flow cyto- metric quantification of the indicated cell populations in the BAL fluid after various HDM-asthma treatments is shown. (C) Quantification of Lin−CD127+T1/ST2+ ILC2 numbers in lung, BAL, and mediastinal lymph nodes (MedLNs) of HDM- or PBS-treated animals is shown. (D) The intracellular cytokine content in HDM-challenged mice is shown. Total lung cell suspensions were stimulated with PMA/ionomycin for 4 h. CD3−CD4−T1ST2+CD127+ cells were gated and analyzed for the indicated cytokines. Values indicate the proportions of cytokine positive cells ± SEM. (E) Cytokine production by lymphocyte populations in lung and BAL fluid is shown. Bars show the absolute numbers of intracellular cytokine-positive cells within the indicated lymphocyte populations. (B, C, E) Data are shown as mean + SEM of n > 4 mice per group and are representative of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t-test. ns: not significant.
  • Innate and innate like lymphocytes in allergic inflammation

    1. 1. Innate and Innate-like Lymphocytes in Allergic Inflammation Jaichat Mekaroonkamol, MD.
    2. 2. outline • Introduction • Innate-like T cells : iNKT – What is iNKT cells – iNKT in asthma • Innate lymphoid cells : ILC2 – What is ILC2 – ILC2 in asthma
    3. 3. Innate vs Adaptive immunity
    4. 4. Innate and Innate-like lymphocytes Innate Immune Cells Adaptive Immune Cells ILCILC Innate-like TInnate-like T Innate-like BInnate-like B
    5. 5. Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
    6. 6. B lymphocyte subsets Abbas. Cellular and Molecular immunology. Seventh Edition
    7. 7. B-1 B cellsB-1 B cells • Self-renewing and preferentially produced in the fetus • Spontaneously produce polyreactive Igs • Low affinity • Response to CHO and Lipid antigen • Natural antibody in mucosa tissue • 5% of total B cell • In mouse: CD5+, Mac-1+ • *In human: CD20+ CD27+ CD43+ CD70-
    8. 8. Marginal Zone B cells • Found in the marginal zone of the spleen – May also be found in the blood • Respond quickly to antigens, e.g. polysaccharides
    9. 9. Characteristics of T, innate-like T and Innate lymphoid cells Conventional T cells Innate-like T cells Innate lymphoid cells TCR (VDJ, rag) Highly diverse Limited diversity No TCR Activate by Peptide Ag on MHC ‘Pattern’ of Ag, cytokines Cytokines, innate receptors Time to response slow fast fast Effector function Cytokines, kill Cytokines, kill Cytokines, kill Examples Th, CTL NKT, γδT NK, ILCs
    10. 10. NKT cells
    11. 11. Natural Killer T (NKT) Cells Unique subset of T cells Function as innate cells, rapidly recognizing pattern of antigens and secrete cytokines to stimulate other cells of innate and adaptive immune system Express characteristics of both T cell and NK cell Semi Variant Non- iNKT iNKT Vα14/Jα18 Vα24/Jα18 Ja18-/- mice lack iNKT cells
    12. 12. Emmanuel Tupin, Yuki Kinjo & Mitchell Kronenberg Nature Reviews Microbiology 5, 405-417 (June 2007) iNKT Antigen Recognition They recognize lipid ! Expanding the repertoire of Ag T cells can sense beyond peptide CD4+ T CD8+ T iNKT
    13. 13. •Potent and specific activator of iNKT cells •Isolated from marine sponge α-Galactosylceramide (α-GalCer) Kawano Science 1997 Tsuji Cell. Mol. Life Sci. 2006 Agelas mauritianus, Kirin Pharmaceutical iNKT ∀α-GalCer loaded CD1d tetramer is now used as gold standard to detect iNKT cells
    14. 14. α-GalCer and analogs GalCer closely related structures forα lipopolysaccharide (LPS) and -proteobacteriaα
    15. 15. Natural ligands -proteobacteriaα •Rickettsiales, Borrelia burgdorferi: Lyme disease, Mycobacterium
    16. 16. Patrick J. Brennan et al. NATURE REVIEWS IMMUNOLOGY, FEBRUARY 2013. iNKT cells during microbial infecti on.
    17. 17. Dingding An et al. Cell 156, 123–13 .
    18. 18. J Allergy Clin Immunol 2013;131:1393-9
    19. 19. But…. • In some circumstances, NKT cells can be activated without the presence of lipid Ag • How could they be activated?
    20. 20. Activation of iNKT cells Patrick J. Brennan et al. NATURE REVIEWS IMMUNOLOGY, FEBRUARY 2013. IL-12, IL-18
    21. 21. Interactions between iNKT cells and other leukocytes. Patrick J. Brennan et al. NATURE REVIEWS IMMUNOLOGY, FEBRUARY 2013.
    22. 22. Borg Nature 2007, Scott-Browne Nat Imm 2007, Bendelac Review 2007, Brennan Nat Rev Imm 2013 AsthmaTumors Infections AutoimmuneTolerance iNKT Cells Function in Diseases •Function as “innate lymphocytes” •Cross-talk with other cells •Bridge innate and adaptive immune responses
    23. 23. iNKT cells in AsthmaiNKT cells in Asthma
    24. 24. Heterogeneity of asthmaHeterogeneity of asthma 1. Antigen-specific Th2 cells 2. Eosinophil
    25. 25. Other pathways beyond Th2 cell and eosinophil?? Other pathways beyond Th2 cell and eosinophil?? 1. Non-allergic asthma is often associated with airway neutrophils rather than eosinophils 2. Treatments that target or eliminate Th2 cells and their cytokines (e.g., anti-IL-4, anti-IL-5 antibodies (Abs) or IL-4 / IL-13 antagonists) have not reduced AHR 3. Most patients with allergic rhinitis and allergen sensitization do not have asthma 4. Elevated levels of non-Th2 cytokines and factors, such as IFNγ, IL-17 and neutrophils are frequently present in patients with severe disease, or with corticosteroid resistant asthma. P Matangkasombut et al. MucosalImmunology, 2009.
    26. 26. iNKT cells and asthma Wild type vsvs Exacerbate /Ameriorate Protective /detrimental vsvs Anti-CD1d blocking Ab iNKT deficient Jα18 -/- CD1d -/- OVA, influenza, ozone, fungus, lipid iNKT adoptive transfer
    27. 27. Everett H. Meyer. Annu. Rev. Med. 2008. 59:281–92
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    29. 29. NKT cells Function in Asthma ILC2 P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009. Chronic lung disease Cytotoxic to regulatory T cells The mechanisms by which iNKT cells function in inducing AHR is unclear
    30. 30. Protein allergens α-GalCer Virus Air pollution Adapted from Busse NEJM 2003 iNKT Lipid Ag Targeting NKT cells for asthma therapy • Direct eliminate iNKT • Paralyzing iNKT cell function • Preventing their activation • CD1d blocking • Modify NKT function (eg. aGalCer and analog) • Eliminate lipid Ag Fungal
    31. 31. iNKT cells in human asthma P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009. At least 13 studies -number of iNKT cells in bronchoalveolar (BAL) fluid, endobronchial biopsies, and sputum samples from patients with asthma -9/13(70%) study shown significant increase -Techniques for identifying iNKT cells were quite varying
    32. 32. Functional of iNKT cells in human P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009. • Functional in vivo studies of iNKT cells are difficult to carry out in humans for safety and ethical reasons • iNKT cells have potent activity, which cannot be assessed simply by counting their numbers in the lung • Non-human primates are closely related to humans in terms of their genome, respiratory physiology, and immunological responses • glycolipids directly activate iNKT cells would result in a similar development of AHR
    33. 33. outline • Innate-like T cells : iNKT – What is iNKT cells – iNKT in asthma • Innate lymphoid cells : ILC2 – What is ILC2 – ILC2 in asthma
    34. 34. Th2 cytokines orchestrate allergic inflammation Kay AB., NEJM 2001, Vol. 344 (1), 30-37. IL-4 IL-5 IL-9 IL-13IL-13 Mucous gland Smooth muscle Mucous gland Smooth muscle Promotes IgE productionPromotes IgE production Promotes tissue eosinophilia and mast cell hyperplasia Promotes tissue eosinophilia and mast cell hyperplasia Promotes mucous production and AHR Promotes mucous production and AHR
    35. 35. Th2 cytokines orchestrate allergic inflammation Kay AB., NEJM 2001, Vol. 344 (1), 30-37. IL-4 IL-5 IL-9 IL-13IL-13 Mucous gland Smooth muscle Mucous gland Smooth muscle Th2 cells are not the only source of type 2 cytokinesTh2 cells are not the only source of type 2 cytokines “Allergic /type 2 inflammation” can occur without adaptive immunity“Allergic /type 2 inflammation” can occur without adaptive immunity
    36. 36. Innate Lymphoid Cells (ILC) secrete Th cytokines Jennifer A. Walker et al. NATURE REVIEWS, 2013.
    37. 37. Innate Lymphoid Cells (ILC) secrete Th cytokines Jennifer A. Walker et al. NATURE REVIEWS, 2013. IL-5, IL-9, IL-13, small amounts of IL-4 IL-5, IL-9, IL-13, small amounts of IL-4 LTα, LTβ,IL-17A, IL- 22 LTα, LTβ,IL-17A, IL- 22 IFNγIFNγ
    38. 38. Jennifer A. Walker et al. NATURE REVIEWS, 2013.
    39. 39. Mary Elizabeth Jones and Yuan Zhuang. V(D)J recombination. Landes Bioscience ,2009.
    40. 40. ID2: transcriptional repressor inhibitor of DNA binding 2 Nature Reviews Cancer 14, 77–91: 2014.
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    43. 43. Jennifer A. Walker et al. NATURE REVIEWS, 2013.
    44. 44. Hergen Spits et al. NATURE REVIEWS IMMUNOLOGY, January 2013
    45. 45. Discovery of ILC2 cells RAG-deficient miceRAG-deficient mice
    46. 46. The Mechanism of V(D)J Recombination Abbas. Cellular and Molecular immunology. Seventh Edition
    47. 47. Adapted from: Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. (without permission) Th2 response
    48. 48. Fallon et al. The Journal of Experimental Medicine: April 10, 2006.
    49. 49. Adapted from: Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. (without permission)
    50. 50. “Nuocytes” Neill Nature April 2010 Saenz Nature April 2010 Mice lacking IL-25 or IL-33 receptors have very few nuocytes and are unable to effectively clear helminth infections
    51. 51. Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. Type 2 Innate Lymphoid Cells (ILC2)
    52. 52. Adapted from: Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013. (without permission)
    53. 53. Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013.
    54. 54. ILC2 in human also produce IL-13 in response to IL-25, IL-33ILC2 in human also produce IL-13 in response to IL-25, IL-33 Mjosberg et al. Nat Imm, 2011
    55. 55. Innate type 2 cells in type 2 immune responsesInnate type 2 cells in type 2 immune responses Neill, Trends in Parasitology, 2011 Spits, Nature Rev Imm, 2013 Moro, Nature, 2010 Neill, Nature, 2010 Saenz, Nature, 2010 Price, PNAS, 2010 IL-33: ST2 IL-25: IL17RB
    56. 56. ILC2 IN (HETEROGENEITY OF) ASTHMA •GWAS of asthma consistently implicate IL-33 and ST2 (Moffatt MF, Nature, 2007. Moffatt MF, NEJM, 2010. Wan YI, Thorax, 2012) •ILC2 participate in asthma induced by •Virus (Chang Y. Nature Imm 2011) •Glycolipid (Kim HY. JACI 2012) •Protease (Halim TYF. Immunity 2012) •Fungal (Bartemes, JI, 2012) •LTD4 (Doherty, JACI, 2013) Rag-/- IL33R-/- Disease No Disease
    57. 57. Chang YJ., Nature Immunology, 2011
    58. 58. Influenza induces asthma independent of T cells But dependent on ILC2 Influenza induces asthma independent of T cells But dependent on ILC2 ST2/IL-33R Chang YJ., Nature Immunology, 2011
    59. 59. AM produce IL-33, increase ILC2 that produce IL-13 essential for H3N1-induced AHR. AM produce IL-33, increase ILC2 that produce IL-13 essential for H3N1-induced AHR. IL-33IL-33 ILC2 in the lungILC2 in the lung Chang YJ., Nature Immunology, 2011
    60. 60. Influenza induce lung ILC to help repair and remodeling Nature Immunology 12, 1025–1027 (2011)
    61. 61. Influenza induce lung ILC to help repair and remodeling Influenza IL-13 Amphiregulin AHR Airway inflammation Klien, Curr Allergy Asthma Rep, 2013 Monticelli LA. Nat Imm July 2011
    62. 62. Glycolipid induced asthma depends on IL-33/ST2, ILC2 and iNKT cells (independent of adaptive immunity) -lipids found in microbes, pollen allergens Kim HY. JACI Jan 2012
    63. 63. Lipid recognitionLipid recognition + Kim HY. JACI Jan 2012
    64. 64. NKT cells Function in Asthma ILC2 P Matangkasombut et al. Mucosal Immunology, SEPTEMBER 2009.
    65. 65. Halim T. Immunity March 2012 •Identified and detailed characterized lung natural helper cells •Use protease allergen (papain) model •Protease are important component of many allergens eg. house dust mites
    66. 66. ILC2-/-ILC2-/- ILC2 are required for papain induced airway inflammationILC2 are required for papain induced airway inflammation Similar data on BAL eosinophils, IL-5, IL-13, mucous productionSimilar data on BAL eosinophils, IL-5, IL-13, mucous production Halim T. Immunity March 2012
    67. 67. ILC2 in fungal induced asthma modelILC2 in fungal induced asthma model Bartemes, JI, 2012 Alternaria induces IL-33 in BAL And induces airway inflammation independent of adaptive immunity but dependent on ST2
    68. 68. LTD4 potentiate Alternaria induced asthma through ILC2 cysLT1R LTD4 potentiate Alternaria induced asthma through ILC2 cysLT1R • Lung ILC2 express cysLT1R • LTD4 induce ILC2 to produce IL-4,IL-5,IL-13 (inhibited by montel ukast) • LTD4 potentiated Alternaria induced eosinophilia and ILC2 acc umulation and proliferation Doherty, D. Broide JACI 2013 ILC2ILC2 LTD4 cysLT1R IL-4, IL-5, IL-13 Montelukast Alternaria
    69. 69. •ILC2 express ALX/FPR2 (receptor for Lipoxin A4) •Lipoxin A4 can suppress IL-13 production by ILC2 in response to PGD2 +/- IL-25/IL-33 Stokes Peebles, Sci Transl Med, 2013
    70. 70. Stokes Peebles, Sci Transl Med, 2013
    71. 71. ILC2 in the lungs M2 AM NKT Pneumocyte type2 Hardman, EJI, 2013 Chang, Nat Imm, 2011 Gorski, PlosPathogen, 2013 Nussbaum, Nature, 2013 Eosinophils homeostasisLTD4 Barnig, Sci Tran Med, 2013 Doherty, JACI, 2013. Barrett, JACI, 2013 ProteaseProtease GlycolipidGlycolipid Montelukast FungusFungusVirusVirus AllergensAllergens remodeling Lipoxin A4 PGD2 Paula Licona-Limón et al. NATURE IMMUNOLOGY, JUNE 2013.
    72. 72. Conclusion • Mechanism of allergic inflammation beyond Th2 • Roles of ILC2 & iNKT cells • Importance of innate and innate-like immunity • Broader ways to initiate and activate “ allergic inflammation” • Novel drug targets for allergic inflammation

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