Qingqing Wang （王青青） Institute of immunology, ZJU

wqq@zju.edu.cn

固有免疫Innate immunity

Contents

• Innate immunity

• Components of the innate immune system

• PAMPs and PRRs

• Innate immune response

2

Innate ImmunityFirst Line of Defense

Characteristics:- rapid- does not generate immunologic memory- dependent upon germline encoded receptors recognizing structures

common to many pathogens

4

Characteristics of Innate and Adaptive Immunity

Characteristics of Innate and Adaptive Immunity

Antigen independent

pattern recognition

No time lag

Not antigen specific

No memory

Antigen dependent

TCR/BCR

A lag period

Antigen specific

Memory

Innate Immunity Adaptive Immunity

Functions

• Innate immunity is the initial response to microbes that prevents, controls, or eliminates infection of the host by many microbes.

• Innate immune mechanisms recognize the products of damaged and dead host cells and serve to eliminate these cells and to initiate the process of tissue repair.

• Innate immunity to microbes stimulates adaptive immune responses and can influence the nature of the adaptive responses to make them optimally effective against different types of microbes.

6

Innate Immune System

1. Physical, chemical ， microbial barriers

- physical barriers include skin and mucus membranes (epithelial)

- chemical barriers include stomach acidity, secreted anti-microbial peptides, defensins ( 防御素 ), cathelicidins ( 抗菌肽 )

- Normal bacterial flora

2. Cells

- macrophages, neutrophils, DC, NK, B1, T, NKT…

3. Blood proteins

- Complement and mediators of inflammation

4. Cytokines

7

8

Barrier

Skin-mucosal barrierPhysical barrier Chemical barrierBiological barrier

Blood brain barrierBlood placental barrier

Epithelial defense mechanisms

10

11

12

表皮 Epidermis of skin

Gut epithelium

13

防水脂质层板层小体：防御素和抗菌肽

What happens when the physical and chemical barriers are breached?

14

Leukocyte Players of Innate Immune Responses

15

Innate-like lymphocytes （ ILLs ）- Unique, minor subsets of T and B lymphocytes that undergo receptor gene

rearrangements to generate receptor diversity (unlike NK cells)

- These subsets express limited receptor diversity, utilizing only a small number of receptor gene segments

- Tend to found in specific locations in the body, usually sites that encounter exogenous antigens or pathogens

16

NK T cell

Distribution

bone marrow, liver, thymus,

spleen, peripheral blood, lymph node

NK T ＜ 1% in blood T cells ； NK T in liver (mouse 30% ， human 4% ）

NK T cell

1, cytokine:

IL-4, IFN-, IL-12

2, cytotoxity

perforin and FasL/Fas

3, MCP-1, MIP-1

Surface marker ： TCR 、 CD4-CD8- 、 CD8+

distribution ： mucosa of skin 、 intestine 、 lung and genitals

restriction ： non-classical MHC molecules Ⅰrestriction

classification ： epidermis T cell

systemic T cell

T 细胞

T cell ： function

Anti-infection

Immune surveilence

Immune regulation

Immune tolerance

Anti-tumor

B1 cell

• B1 cell mainly located in abdomen cavity, thorax and intestines

• B1 cell recognize LPS and capsular polysaccharide

• IgM antibody• No class switch and immune memory

1. Two types of phagocytes(1) Neutrophil granulocytes

1. Two types of phagocytes(1) Neutrophil granulocytes

phagocytosis, intracellular killing, inflammation and tissue damage

characteristic nucleus, cytoplasm

granules and CD67 membrane marker.

(2) Monocytes and macrophages(2) Monocytes and macrophages

phagocytosis, intracellular and extra-cellular killing, tissue repair, antigen presentation for specific immune response

characteristic nucleus and CD14 membrane marker.

（ 2 ） monocyte/macrophage

macrophage

• cDC

myeloid DC, conventional DC

• pDC (plasmacytoid DC)

TLR7,9 type I interferon

Dendritic cells, DC

Innate-like lymphocytes （ ILLs ）- Unique, minor subsets of T and B lymphocytes that undergo receptor gene

rearrangements to generate receptor diversity (unlike NK cells)

- These subsets express limited receptor diversity, utilizing only a small number of receptor gene segments

- Tend to found in specific locations in the body, usually sites that encounter exogenous antigens or pathogens

30

Complement system ：

31

32

Cytokines of innate immunity

33

34

TNF

35

IL-1

Innate Immune Receptors

Innate immune receptors are not clonally distributed

Binding of receptors results in rapid response

Innate immune receptors mediate three functions:

- phagocytic receptors to stimulate pathogen uptake

- chemotactic receptors that guide phagocytes to site of infection

- stimulate production of effector molecules and cytokines that induce

innate responses and also influence downstream adaptive immune responses

36

Most microorganisms express repeating patterns of molecular structures termed Pathogen Associated Molecular Patterns (PAMPs)

Endogenous molecules that are produced by or released from damaged and dying cells are called Damage-Associated Molecular Patterns (DAMPs)

Innate immune system has evolved mechanisms capable of recognizing these repeating patterns termed Pattern Recognition Receptors (PRRs)

Pathogen Recognition

37

38

免疫识别的危险模式理论Danger signalDanger Theory

Polly Matzinger 理论免疫学家

PAMPs

DAMPs

41

Structure of bacteria

pilin for

adhesion

flagellum for motion

shape and rigidity

DNA

42

43

Shared features of bacteria: cell walls

peptidoglycanpeptidoglycan

teichoic acid

proteins

lipopolysaccharide proteins

peptidoglycanpeptidoglycan

IM

OM

胞壁酸

Peptidoglycan 肽聚糖

peptidoglycanpeptidoglycan

teichoic acidproteins

44

N- 乙酰氨基葡糖和 N- 乙酰胞壁酸交替的杂多糖

45

Lipopolysaccharide 脂多糖

peptidoglycanpeptidoglycan

IM

OM

lipid Alipid A 类脂质 A

46

Flagellin 鞭毛

钩状体 丝状体

Bacterial DNA

Bacteria• Frequent CG sequences• Cytosine not methylated

Mammals• Rare CG sequences• Cytosine in CG is methylated

47

Viruses and double-stranded RNA

• Some viruses have dsRNA genomes (rare)

• Many more viruses have single-stranded RNA genomes but produce dsRNA during replication and mRNA synthesis

48

Engagement of innate immunity

• Recognition of organisms based on conserved patterns– Lipopolysaccharide (gm-)– Peptidoglycan (most bacteria)– Flagella (many bacteria)– Unmethylated CpG in DNA– Single-stranded RNA (viral)

49

Examples of Pattern Recognition Receptors (PRRs) :- Mannose-Binding Lectin (MBL)- Macrophage Mannose Receptor- Scavenger Receptors- Toll-like Receptors (TLRs)- RIG-I like Receptors (RLRs) - Nod-like Receptors (NLRs)

Pathogen Recognition

50

51

Toll-like receptors

single strandedsingle stranded RNARNA

TLR8TLR8

Toll-Like Receptors (TLRs)

Cellular Localization:- Lysosomal localization (i.e. subcellular) of TLR-3 and TLR7,8,9- TLR-3 and 7,8,9 recognize viral/bacterial nucleic acids- lysosomal expression isolates pathogen nucleic acid recognition away from

potential cross-reaction with host mammalian nucleic acid motifs

TNF

IFN

52

53

RLRs ： Retinoic Acid-Inducible Gene-I-like Receptors

RIG-I , Retinoic Acid-Inducible Gene-1

MDA5, Melanoma Differentiation-Associated Gene 5 CARD-containing; melanoma growth-suppressive properties

LGP2, Laboratory of Genetics and Physiology 2 DExH/D-box motif and a carboxy-terminal helicase domain

RD

54

RIG-I Like Receptors (RLRs)

55

56

57

Nod-like receptors (NLRs)

• Nucleotide-binding and oligomerization domain (NOD)-like receptors

• Cytoplasmic proteins

• At least 22 members

• Recognize microbial components

• Work synergistically with TLR

MDP：胞壁酰二肽iE-DAP： γ 谷氨酰基二氨基庚二酸

NOD-I Like Receptors (NLRs)

NLRs are cytoplasmic bacterial sensors 14 NLRPs activate IL-1β Inflammasome IPAF/NLRC4 NLRP3 NLRP1

58

Cell-associated PRRs

59

Other cell-associated PRRs

60

二酰基甘油酯

Soluble recognition and effector molecules

61

Summary and Review of Innate Immune Responses

62

Correlation between innate immunity and adaptive immunity

• Initiation of adaptive immunity

• Influence on type of adaptive immunity

• Participate in effect phase of adaptive immunity

NK cells

NK cells were defined initially

by their ability to lyse certain

tumor cell lines and virally

infected cells without prior

immunization, and so mediate

a form of innate (or natural)

immunity that is termed

“natural killing”.

NK cells were defined initially

by their ability to lyse certain

tumor cell lines and virally

infected cells without prior

immunization, and so mediate

a form of innate (or natural)

immunity that is termed

“natural killing”.

Surface markers

1. CD56

2. CD16(FcγR ) ADCCⅢ

3. FcR

NK cells are generally TCR- , mIg-, CD16+,

CD56+.

66

Natural killer cells

• Development

• Receptors

• Function

67

Activation of NK cells is the net effect of inhibitory and activating signals

NK receptors

68

The receptors associated with activation or inhibition

1.The receptors recognizing MHC moleculesⅠ(1) Killer immunoglobulin-like receptor, KIR KIR 2DL ITIM inhibitory receptor KIR 2DS binding with DAP-12( ITAM) activating receptor KIR 3DL ITIM inhibitory receptor KIR 3DS binding with DAP-12( ITAM) activating receptor Intracellular signaling pathways coordinate inhibitory and

activating signals

(2) Killer lectin-like receptor, KLR

CD94

NKG2A ITIM inhibitory receptor

CD94

NKG2C binding with DAP-12( ITAM)

activating receptor

NK receptors: ‘Defense is the best offense”

While both KIRs and KLRs sense the presence (absence) of MHC class I molecules, activating as well as inhibitory receptors are found in both families of receptors.

- The KIRs are subdivided according to the number of immunoglobulin-like domains (2 or 3 domains) and the length of their cytoplasmic tail: Short tail = activating receptorsLong tail = inhibitory receptors

- The KLR are heterodimers of CD94 associated with a NKG2 molecule. Six distinct NKG2 isoforms exist in humans.NKG2C/CD94 = activating receptorNKG2A/CD94 = inhibitory receptorNKG2B/CD94 = inhibitory receptorNKG2D homodimer = activating receptor.

72

(3) The significance of KIR and KLR

NK cells are prevented from killing host cells

because they recognize the host class MHC Ⅰmolecules. Thus, they only attack cells whose

class MHC molecules are lost or altered, as occurs Ⅰfrequently in malignancy or viral infection.

2. The receptors recognizing non-MHC Ⅰmolecules

(1) NKG2D binding with DAP-10 ( ITAM) activating receptor ligand: M C A/B (on some tumor cells)Ⅰ

(2) natural cytotoxicity receptor, NCR NKp46,NKp30 binding with CD3( ) NKp44 expressed on activated NK cells binding with DAP-12 ( ITAM) activating receptor

75

Functions of NK cells

1. Anti-infection provide an early innate barrier to infection by

eliminating infected host cells

2. Anti-tumor kill tumor cells directly: perforin, granzymes, ADCC

3. Immunoregulation Activated NK cells produce cytokines such as IFN-,

TNF-, G-CSF. These cytokines can activate T cells.

Functions

77

The mechanism of cytotoxicity

1. Perforin / granzyme pathway

2. Fas / FasL pathway

3. TNFα / TNFR-1

target cell apoptosis

Release of cytotoxic granules at the site of contact with infected cells

- First contact between a CTL or NK cell with infected cells is via non-specific binding of adhesion molecules (LFA-1 (blue) on T and NK cells with ICAM-1 or ICAM-2 (brown) on target cells). This makes a channel between the target and the cytotoxic cell. - Specific antigen/MHC class I recognition by TCR on CTL, or engagement of the NK’s natural cytotoxic receptors (NCR) (green) by non-MHC ligands (orange) on the surface of the target cell. This results in a polarization of the cell: the actin cytoskeleton (green staining in the immunofluorescence microscopy) at the site of contact is reorganized as to aligning the microtubule-organizing center (MTOC), as well as the secretory apparatus, including the Golgi (GA). The GA-derived lytic granules (stained in red in the photomicrograph) are specifically directed onto the target cell.- The content of the granules is directly released onto the target cell.

Target cell

NK cell

Why are NK cells prominent tumor killers?

- The regression of transplanted tumors in a normal mouse model (blue line) is largely due to the action of CTLs recognizing tumor antigens presented on MHC class I (right panel). Albeit the presence of NK cells, this regression is absent in nude mice (red line) in which CTLs do not develop. -Tumor variants that express low levels of MHC class I become susceptible to NK cells, especially in nude mice (have higher levels of NK cells than wild type mice). Thus tumors that are sensitive to NK killing grow less well in nude than normal mice (central panel).- Transfection of MHC class I genes resulting in high expression of this protein restores NK cell resistance but susceptibility to CTL in normal mice (left panel; blue line).

Similar to many pathogens, tumor cells can escape the adaptive immune system, by downregulating the expression of MHC class I.This makes them more susceptible to NK cells.

Thanks for your attention!

If you have any questions, please feel free to contact me: wqq@zju.edu.cn