1. INNATE IMMUNITY: ANTIVIRAL STATE, KILLER CELLS, THE COMPLEMENT SYSTEM
3RD SEMINAR
INNATE IMMUNITY: ANTIVIRAL STATE, KILLER CELLS, THE COMPLEMENT SYSTEM

2. DANGER SIGNALS ARE TRANSLATED TO CYTOKINE SECRETION THROUGH VARIOUS MOLECULAR SENSORS IN DC SUBTYPES4 6 6 2 1 1 5 10 3 7 9 7 8
RLR
NLR
RLR
Plasmacytoid DC
Conventional DC
IL-1β
IL-12/23
IL-10
TLR1 – bacterial lipoprotein (together with TLR2)
TLR2 – bacterial lipoprotein, peptidoglycane, lipoteicholic acid
(heteromer with TLR1 and TLR6)
TLR3 – viral dsRNA
TLR4 – bacterial LPS
TLR5 – bacterial flagellin
TLR6 – bacterial lipoprotein (together with TLR2)
TLR7 – viral ssRNA
TLR8 – viral ssRNA
TLR9 – unmethylated CpG DNA
TLR10 – modified viral nucleotides
NLRs – microbial products, DAMPs
RLRs – viral dsRNA
IFNα IFNβ

3. THE TYPE I INTERFERON RESPONSE: ANTIVIRAL STATE
plasmacytoid dendritic cells
Plasmacytoid dendritic cells (pDCs) produce 1000x more type I interferon than other cells (Natural Interferon Producing Cells – NIPC)
After viral infection they are accumulated at the T cell zone of the lymph nodes

4. VIRUS-INDUCED TYPE I INTERFERON PRODUCTION
paracrine
autocrine
Infected cell
subtypes
IFN-
IFN-
IFN response
IRF-3
IRF-7
Virus
NFB
AP-1
Type I IFN receptor
IRF: interferon regulatory factor

5. INTERFERON EFFECTOR PATHWAYS induction of the „antiviral state”
1. Mx GTPase pathway
block viral transcription
2. 2',5'-oligoadenylate-synthetase (OAS)-directed Ribonuclease L pathway
degrade viral RNA
3. Protein kinase R (PKR) pathway (Ser/Thr kinase, dsRNA- dependent)
inhibit translation
4. ISG15 ubiquitin-like pathway
modify protein function
CONTROL ALL STEPS OF VIRAL REPLICATION

6. MULTIPLE EFFECTS OF TYPE I INTERFERONS
TRIF
TANK
IKKε
TBK1
IRF-3
TRAM
TLR3
TLR4
MyD88
IRF-5
TLR7
TLR8
TLR9
IFN-β, IFN-α
RIG-1
Stimulation of Ig-production
in B-cells
Type I interferon receptor
IRF-7
Increased citotoxicity and proliferation of NK-cells
Activation of - and γδ T-cells
Increased antigen presentation
in myeloid dendritic cells
IRAK-1
TRAF-6

7. EFFECTOR MECHANISMS OF INNATE IMMUNITY
KILLER CELLS
COMPLEMENT SYSTEM
PHAGOCYTIC CELLS

8. NK CELLS Similar functions to cytotoxic T cells but:
larger than lymphocytes
no rearranged antigen-specific receptors
contain large cytoplasmic granules
respond fast, circulate in a partly activated state

9. RECOGNITION AND KILLING BY NK CELLS
KIR
KAR
Contents of lytic granules:
Perforin: forming pores in the target cell membrane  lysis
Granzyme: inducing apoptosis in the target cell

10. NATURAL KILLER CELL ACTIVATION
NK-CELLS
Virus-infected
cell
PRR
RECOGNITION
ACTIVATION
Lysis of infected cell
RECOGNITION OF ALTERED HOST CELLS
Kinetics of the activity of the complement system and NK cells in virus infection
IFN
IL-12
Complement system
NK-cells
days
Relative level/activity

11. EFFECTOR MECHANISMS OF INNATE IMMUNITY
KILLER CELLS
COMPLEMENT SYSTEM
PHAGOCYTIC CELLS

12. THE COMPLEMENT SYSTEM
The complement system is a set of plasma proteins that act in a cascade to attack and kill extracellular pathogens.
Approximately 30 components:
activating molecules
regulator factors
complement receptors
membrane proteins which inhibit the lysis of host cells
Most of the complement proteins and glycoproteins are produced in the liver in an inactive form (zymogen). Activation is induced by proteolytic cleavage.

13. AMPLIFICATION OF THE COMPLEMENT CASCADE
limited
proteolysis
inactive precursors
enzyme
activating surface
Activating surface needed!

14. ACTIVATION OF THE COMPLEMENT SYSTEM
Clearence of Immune complexes

15. THE CLASSICAL PATHWAY

16. THE C1 COMPLEX
Collagen „legs”
Gobular „heads”
C1 is always present in serum but it requires an activating surface for activation
Low affinity binding to the Fc region of antibody  conformational change  activation

17. ACTIVATION OF THE C1 COMPLEX

18. THE CLASSICAL PATHWAY: FIXATION OF COMPLEMENT, GENERATION OF C3b BY THE CLASSICAL C3 CONVERTASE
When soluble pentameric IgM in the 'planar' conformation establishes multipoint binding to antigens on a pathogen surface, it adopts the 'staple' conformation and exposes its binding sites for the C1q component of C1. Activated C1 then cleaves C2 and C4, and the C2a and C4b fragments form the classical C3 convertase on the pathogen surface. Conversion of C3 to C3b leads to the attachment of C3b to the pathogen surface and the recruitment of effector functions (i.e. opsonisation). C3a recruits phagocytic cells to the site of infection.

19. THE MANNAN-BINDING LECTIN PATHWAY

20. GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES
Eukariotic cells
Prokariotic cells
galactose
glucoseamine
mannose (polymer = mannan)
neuraminic acid
(sialic acid)

21. MANNOSE-BINDING LECTIN (MBL) PATHWAY
MBL: part of the collectin family
similar structure to C1 complex, MASP-1,2 ~ C1r,s
binds mannose and similar sugar molecules on the surface of bacteria, fungi, protozoa and viruses  conformational change  cleavage of C2 and C4 molecules
MASP = MBL-associated serin protease

22. ACTIVATION OF THE MBL COMPLEX
The literature sometimes refers to the bigger fragment of C2 as C2a but it is more consequent if we use ‚b’ for the bigger fragments of all complement molecules

23. THE ALTERNATIVE PATHWAY

24. C3b can derive from classical or the lectin pathway too
Alternative pathway is instantly inactivated on eukaryotic cell surfaces (in the presence of sialic acid molecules)
In the plasma close to a microbial surface the thioester bond of C3 spontaneously hydrolyzes at low frequency. This activates the C3, which then binds factor B. Cleavage of B by the serine protease factor D produces a soluble C3 convertase, called iC3Bb, which then activates C3 molecules by cleavage into C3b and C3a. In this complex the Bb fragment of factor B provides the protease activity to cleave C3, and the C3b fragment of C3 locates the enzyme to the pathogen's surface.

25. THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM
C3 proteis have one of the highest levels in the serum: 1.2 mg / ml
Strong covalent binding Complement fixation
( molecules/ml)

26. C3 convertase + C3b = C5 convertase
(C4bC2bC3b)
Figure 2.12 Complement component C5 is cleaved by C5 convertase to give a soluble active C5b fragment.
The C5 convertase of the alternative pathway consists of two molecules of C3b and one of Bb (C3b2Bb). C5 binds to the C3b component of the convertase and is cleaved into fragments C5a and C5b, of which C5b initiates the assembly of the terminal complement components to form the membrane-attack complex.
The classical and alternative C3 convertase is different in structure but common in function

27. MEMBRANE ATTACK COMPLEX (MAC = C5b-C9n)
MACs in the cell membrane
Pore formation  osmotic lysis of pathogens

28. COMPLEMENT ACTIVATION
SUMMARY

29. C3 CONVERTASE C3b Antigen-antibody complex Mannose Pathogen surface
COMPLEMENT SYSTEM
CLASSICAL PATHWAY
MB-LECTIN PATHWAY
ALTERNATIVE PATHWAY
Antigen-antibody
complex
Mannose
Pathogen surface
MBL
MASP-1/MASP-2
Serin protease
C4, C2 C3
B, D
C1q, C1r, C1s
Serin protease
C4, C2
C4a*
C3a, C5a
C3 CONVERTASE
C3b
Terminal C5b – C9
Inflammatory peptide mediators
Phagocyte recruitment
Opsonization
Binding to phagocyte CR
Immune complex removal
MAC
Pathogen/cell lysis

30. THE ANAPHYLATOXINS: C3a, C4a, C5a

31. OPSONIZATION COMPLEMENT-MEDIATED PHAGOCYTOSIS

32. MOVIES: ACTIVATION VIA THE CLASSICAL PATHWAY

33. REGULATION OF THE COMPLEMENT SYSTEM

34. DEFICIENCIES OF COMPLEMENT COMPONENTS AND REGULATORS
Deficient complement protein
Effects of deficiency
C1, C2, C4 C3
Immune-complex diseases (similar to SLE), susceptibility to pyogenic infections
MAC, alternative pathway components
Susceptibility to Neisserial infections
C1INH
Hereditary angioneurotic edema (HANE)
DAF (CD55), MIRL (CD59)
Paroxysmal nocturnal hemoglobinuria (PNH)

35. HEREDITARY ANGIONEUROTIC EDEMA (HANE) (HEREDITARY C1INH DEFECT)
Inhibition by C1INH in many steps
bradykinin and C2-kinin: enhance the permeability of postcapillar venules  edema
C1 is always active without activating surface because plasmin is always active
Main symptoms:
swellings of skin, guts, respiratory tracts
serious acute abdominal pain, vomiting
larynx swelling – suffocation, may cause death
Treatment:
iv C1INH, FFP, steroid
kallikrein and bradykinin receptor antagonists
FFP= Fresh Frozen Plasma
Children with symptoms of HANE

36. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
Acquired clonal mutation of PIG-A gene in myeloid progenitors – no GPI-enchored proteins in the cell membrane of affected cells (rbc, plt, wbc)
CD59 and CD55 complement regulatory proteins are GPI-enchored proteins
No CD59 and/or CD55  PNH patients are highly susceptible to complement-mediated lysis
The lysis of red blood cells leads to high levels of hemoglobins in the blood that appears in the urine (hemoglobinuria)
Elevated levels of TF derived from complement-damaged leukocytes cause thromboses
PIG-A = Phosphatidylinositol N-acetylglucosaminyltransferase subunit A
GPI= glycosylphosphatidylinositol
Paroxysmal = sudden attacks
Nocturnal = occuring at night

37. Change in the colour of urine samples taken from PNH patient during the day
Only minority of patients have this symptom.

38. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
SYMPTOMS
THERAPY
Haemolytic anaemia and
associated symptoms
Haemoglobin and its products in the urine
Thrombosis:
brain veins,
mesentheric veins,
hepatic veins (Budd-Chiari-syndrome)
May transform to leukemia or other bone marrow diseases
Specific th.: eculizumab (Soliris) = anti-C5 monoclonal antibody
Curative th.: bone marrow transplantation
Alternative th.: steroids (general immunosuppression)
Anticoagulants: s.c. heparin  p.o. kumarin
Iron replacement
Transfusion (filtered-irradiated blood)