Download 5th seminar MHC, high throughput, hypesrensitivity (2016)

Functional investigations of the cells
of the immune system (3rd part)
Further analytical methods based on antigenantibody interactions
MHC typing
Examination of the hypersensitivity reactions
(allergy and other hypersensitivity reactions)
(The pro5.exe (4414KB) is an executable animation file which can be started from the “MHC multimer” slide of this slide show)
(review/revise)
Receptor crosslinking
(immediate)
phosphorilation steps
- Western blot
- Bead array
(seconds-minutes)
Antigen receptors (TCR, BCR), and different
other receptors (e.g. cytokine receptors)ic Ca2+ increase
- FACS, microscopy
Gene activation
- RT-PCR
Cytokine synthesis - IC cytometry
Cytokine secretion
Cell-cycle/apoptosis
Lymphocyte activation
Cell division
- ELISA, ELISPOT
- DNA content
- IN antigens
- 3H-thymidine, CFSE, MTT
The examination often requires specific
Ag-Ab reactions
(review/revise)
THE SIGNAL INTENSITY IN THE METHODS
BASED ON ANTIGEN-ANTIBODY INTERACTIONS
IS PROPORTIONAL TO THE AMOUNT OF
ANTIGEN PRESENT
Enzyme activity is measured by the color
reaction due to conversion of substrate in ELISA
Similar principle applies to many other antibody-based
detection methods
High throughput screening methods
based on antigen-specific antibodies
(can be considered as proteomic methods)
CYTOKINE ARRAY
Simultaneous detection of multiple cytokines
multiple antigen specific antibodies bound to a membrane
(a kind of protein binding membrane as in WB)
labeled antibody mixture
(+)
IL-2
IL-4
the sample which
contains cytokines
…
…
MIP3β
IFN
…
(
Disadvantage: Relatively large sample volume is
needed to cover the surface of the membrane
-)
“Sandwich”
Dendritic cells
were activated either
by CD40L
or by the combination of
CD40L+SLAM molecules
Réthi és mtsi. 2006
Any kind of antigen and modified antigens
can be also examined:
E.g.: Signalling of untreated and UV light irradiated cells
examination with a “phosphoprotein intracellular signalling array kit”
 chemoluminescence / densitometry
(+)
(+)
(+)
(-)
Protein array
Phosphoprotein array  signalling pathways
High throughput cytokine examinations by
flow cytometry
(proteomics)
It can be performed with small
volumes (5-20ml) also
Cytometric multiplex bead array (CBA)
The cytokine capture beads are identified by their colour intensities
IL-2
cytokine array
CBA
IL-4
IL-8
…
…
IL-12
IL-23
IL-17
…
…
Cytometric / multiplex bead array (CBA)
Comparison of CBA with the sandwich ELISA
Sandwich ELISA
CBA
IL-2 „reporter” antibody
IL-2
bead with
capture antibodies
IL-4
IL-2
capture antibodies
IFN
sample
cytokine
concentration
The cytokine
concentration is
proportional with the
color intensity of the
reporter antibody
TNFα IL-6 IL-8
IL-12 IL-10 IL-1β
500pg/ml
250pg/ml
125pg/ml
62pg/ml
31pg/ml
16pg/ml
cytokine standard serial dilution
8pg/ml
0
Cell signalling and protein modifications can be also
examined by cytometric bead arrays (similarly to the
protein arrays)
•
•
•
•
phosophoproteins
ubiquitinylation
acetylation
etc.
Examination of the antigen-specific T-cells
e.g.: monitoring the efficiency of a
vaccination
antigen
specific
T-cell
T-cell clones with identical
T-cell antigen receptors
immunisation
The specificity of the T-cell receptors (TCR) should be identified
Antigen receptors can be identified with the antigen.
TCR can be identified with the ligand:
labelled MHC-peptid complex
The interaction between the TCR and the MHC is too low affinity
MHC
T-cell receptors
T-cell
A single MHC molecule
can only temporarily
bind to a single TCR
Multimeric MHC-peptid complex can have appropriate avidity for efficient binding
MHC pentamer technics
one unit of the pentamer
peptide
MHC
self assembling
coiled-coil-domain
fluorescent label
composed
pentamer
MHC pentamer binding to T-cell
MHC pentamer
T-cell receptors
MHC-peptide oligomers
have high avidity
interactions with the
specific TCRs
MHC multimers could help revealing the
antigen specific T cell numbers, which
is useful in the estimation of the
efficiency of new vaccination or T-cell
therapeutic methods
peptide-specific
T-cell
Launch pro5.exe
short animation
MHC dextramers – MHC/peptie complexes can be bound
to dextrane polimers to form multimeric complexes
MHC tetramers – avidin has tetrameric biotin binding domains,
so it can bind 4 biotinylated MHC/peptide complexes
https://www.benaroyaresearch.org/what-is-bri/scientists-andlaboratories/core-labs/tetramer-biomarker
EBV BZLF-1 (RAKFKQLL/
HLA-B*0801)-specific T-cells
(carriers: 90-95% of the normal healthy
human population)
MHC multimeric staining
CMV-specific T-cells in a
healthy HLA-A2 donor
Healthy people can have
high number of
herpesvirus specific T
cells. Latent infections
keep the number of
antigen specific
memory/effector cells
high.
Influenza epitope (GILGFVFTL/
HLA-A0201)-specific (memory)
T-cells in a healthy donor
You should know the MHC specificity of
the T cells to perform the examinations!
(MHC restriction of the T cells)
allél
sequence
Tumour (associated) peptide epitops
A*0201
GVLVGVALI
Carcinogenic Embryonic Antigen (CEA) 694-702
A*0201
LLGRNSFEV
p53 261-269
A*0201
LLLLTVLTV
MUC-1 12-20
A*0201
RLLQETELV
HER-2/neu 689-697
A*0201
RMFPNAPYL
Wilm's Tumour (WT1) 126-134
A*0201
SLLMWITQV
NY-ESO-1 157-165
A*0201
STAPPVHNV
MUC-1 950-958
A*0201
VISNDVCAQV
Prostate Specific Antigen-1 (PSA-1) 154-163
A*0201
VLQELNVTV
Leukocyte Proteinase-3 (Wegener's autoantigen) 169-177
A*0201
VLYRYGSFSV
gp100 (pmel17) 476-485
A*0201
YLEPGPVTA
gp100 (pmel17) 280-288
A*0201
YLSGANLNL
Carcinogenic Embryonic Antigen (CEA) 571-579
A*0201
KVLEYVIKV
MAGEA1 278-286
A*0201
KVAELVHFL
MAGEA3 112-120
A*0201
KTWGQYWQV
gp100 (pmel17) 154-162
A*0201
HLSTAFARV
G250 (renal cell carcinoma) 217-225
A*0201
ILAKFLHWL
Telomerase 540-548
Examples of some available human
MHC/peptide pentamers
You should know the HLA type of the
investigated person
allele
sequence
EBV epitope
A*0201
CLGGLLTMV
EBV LMP-2 426-434
A*0201
GLCTLVAML
EBV BMLF-1 259-267
A*1101
IVTDFSVIK
EBV EBNA-4 416-424
A*2402
TYGPVFMCL
EBV LMP-2 419-427
B*0702
RPPIFIRRL
EBV EBNA-3A 247-255
B*0801
FLRGRAYGL
EBV EBNA-3A 193-201
B*0801
RAKFKQLL
EBV BZLF-1 190-197
B*3501
HPVGEADYFEY
EBV EBNA-1 407-417
A*0201
ILHNGAYSL
HER-2/neu 435-443
allele
sequence
A*0201
IMDQVPFSV
gp100 (pmel17) 209-217
Influenza A epitope
A*0101
CTELKLSDY
Influenza A (PR8) NP 44-52
A*0201
KIFGSLAFL
HER-2/neu 348-356
A*0201
GILGFVFTL
Influenza A MP 58-66
A*0201
LMLGEFLKL
Survivin 96-104
A*0301
ILRGSVAHK
Influenza A (PR8) NP 265-274
A*0201
ALQPGTALL
Prostate Stem Cell Antigen (PSCA) 14-22
A*0201
CMTWNQMNL
Wilm's Tumour (WT1) 235-243
A*0201
ELAGIGILTV
MelanA / MART 26-35
allele
sequence
HIV epitope
A*0201
FLTPKKLQCV
Prostate Specific Antigen-1 (PSA-1) 141-150
A*0201
ILKEPVHGV
HIV-1 RT 476-484
A*0201
GLYDGMEHL
MAGEA-10 254-262
A*0201
KLTPLCVTL
HIV-1 env gp120 90-98
A*0301
KQSSKALQR
bcr-abl 210 kD fusion protein 21-29
A*0201
SLYNTVATL
HIV-1 gag p17 76-84
A*0301
ATGFKQSSK
bcr-abl 210 kD fusion protein 259-269
A*0201
TLNAWVKVV
HIV-1 gag p24 19-27
A*0301
ALLAVGATK
gp100 (pmel17) 17-25
A*0301
QVPLRPMTYK
HIV-1 nef 73-82
A*2402
VYGFVRACL
Telomerase reverse transcriptase (hTRT) 461-469
A*0301
RLRPGGKKK
HIV-1 gag p17 19-27
A*2402
TYLPTNASL
HER-2/neu 63-71
A*2402
RYLKDQQLL
HIV-1 gag gp41 67-75
A*2402
TYACFVSNL
Carcinogenic Embryonic Antigen (CEA) 652-660
B*0702
IPRRIRQGL
HIV-1 env gp120 848-856
A*2402
TFPDLESEF
MAGEA3 97-105
B*0702
TPGPGVRYPL
HIV-1 nef 128-137
A*2402
EYLQLVFGI
MAGEA2 156-164
A*2402
CMTWNQMNL
Wilm's Tumour (WT1) 235-243
A*2402
AFLPWHRLF
Tyrosinase 188-196
B*0801
GFKQSSKAL
bcr-abl 210 kD fusion protein 19-27
B*0801
FLKEKGGL
HIV-1 nef 90-97
B*0801
GEIYKRWII
HIV-1 gag p24 261-269
B*2705
KRWIILGLNK
HIV-1 gag p24 265-274
H-2Kd
AMQMLKETI
HIV-1 gag p24 199-207
MHC TYPING
and some transplantation immunology
Natural populations have multiple MHC isotypes and
allelic variants
human MHC antigens  human leukocyte antigens (HLA)  HLA typing
polymorphic MHC I isotypes: HLA-A, HLA-B, HLA-C
polymorphic MHC II isotypes: HLA-DP, HLA-DQ, HLA-DR
laboratory mouse MHC antigens  H-2 antigens  H-2 typing
MHC I: H-2K, H-2D, H-2L
MHC II: H-2A (I-A), H-2E (I-E)
(Laboratory animals are inbred and their MHC types are known)
HLA typing
human polymorphic isotypes:
MHC I: HLA-A, HLA-B, HLA-C
MHC II: HLA-DP, HLA-DQ, HLA-DR
• used in organ/tissue transplantation
Polimorphic isotypes have higher chance to differ between individuals
The most polymorphic isotypes (HLA-B and HLA-DR) could cause most of the
problems in the transplantation
HLA-C is less polymorphic, less immunogenic, so it can have smaller negative
effect on the success of the transplantation
• can have diagnostic value
Significant correlation can be shown between the presence of some HLA allelic
variant and the incidence of diseases. The correlation can be either positive or
negative
(e.g.: autoimmune diseases, hypersensitivity, infections….)
•
as you seen before in connection with the MHC multimers, you can’t use some
examination methods without prior typing the MHC of your subject
Some term in connection with MHC types and transplantation:
autologous – derived from the self/same organism (MHC identical)
syngeneic – have the same MHC alleles (e.g. identical twins, identical mouse strains)
allogeneic – have different MHC alleles (e.g. derived from an other person)
xenogeneic – MHC and other genetic differences between species
(e.g. “blood group” antigenic differences)
Transplant reactions
(antibody or T cell mediated effector functions)
• Hyperacute rejection
Pre-immunised state of the
recipient:
Presence of anti-MHC or anti
blood group (AB0) antibodies
(blood group mismatch)
antibodies bind to the endothelial
cells of the organ
• activation of the complement
system
• FcR mediated reactions
• trombosis
• vascular necrosis
• Acute rejection:
- HLA-A, B, C, DR, DQ, DP
differences
- minor histocompatibility
antigen differences
allogeneic MHC or antigens can
be recognised by T cells:
Direct: T cells recognise allogeneic
MHC expressing cells (CD8+
CTL cytotoxicity)
Indirect: professional self APC
present alloantigens from the
graft (allogeneic MHC proteins
can give rise peptides also)
(CD4+ T cells: cytokine
production, inflammation)
therapy resistant
INFLAMMATION, ORGAN REJECTION
Diseases
(autoimmune)
HLA
frequency
sick
control
SLE
DR3
B8
55
50
20
20
Hiydralazin induced lupus
erythematosus
DR4
73
32
DR3
B8
DR3
DR2
B7
B8
56
43
55
60
37
44
25
20
21
30
24
20
DR4
53
19
DR3
DR3
B8
DR3
B8
DR3
B8
DR3
DR7
B8
DR2
DR3
DR3
B8
B35
B27
DR4
72
49
40
70
46
70
50
79
60
68
88
81
82
75
70
79
95
24
22
21
20
23
20
22
22
15
22
29
20
20
22
15
9
20
Basedow-disease
Chronic active hepatitis
Sclerosis multiplex
Myasthenia gravis
Autoimmune IgA
glomerulonephritis
Type I diabetes
Addison-disease
Sjörgen-syndrome
Coeliakia
Goodpasture-syndrome
IgA defficiency
Dermatitis herpetiformis
De Qervain-thyreoiditis
Reiter-syndrome
Felty-syndrome
HLA allotípusok és betegségek
közötti kapcsolatok
Diseases
Narcolepsy
Bechterew-disease
Adrenogenital syndrome
salt wasting
late form
virilisating form
Psoriasis vulgaris
Idiopathic haemochromatosis
Bechet-disease
Gold induced thrombocytopenia
Gold induced leukopenia
Klinikai immunológia (II. klinikum) (OHVI 1990 szerk.: Szegedi, Gergely, Sipka, Szemere)
Stenszky Valéria: Autoimmun betegségek genetikai vonatkozásai
HLA
frequency
sick
control
DR2
B27
100
89
22
9
Bw47
B14
B5
Cw6
B13
B17
DR7
A3
B51
DR3
DR3
36
57
48
56
24
27
48
76
50
50
47
1
4
10
15
8
8
23
28
11
13
13
HLA typing methods:
The classical immunology method:
Serotyping
Antibodies in the typing sera can recognise the HLA antigens on the cells.
Different sera can be used to react with the isotypes of MHC I or MHC II. The sera can
discriminate between different allelic types (allotypes). Classical complement activation
pathway mediates the lysis of the recognised cells:
If the typing serum don’t react with the cells there is no lysis or membrane damage
The slightly damaged cells can be stained and the degree of damage observed by microscopy can get some “score”
•
Typing sera could be obtained from immunised persons in the past (e.g. women after multiple
pregnancies or persons after blood transfer can be immunised by different MHC alleles)
Monoclonal antibodies are used for the purpose recently.
•
Serum/antibody specificity and new HLA allelic variants are
discussed / exchanged on international workshops
•
The quantity of the well characterised typing seran is limited
 typing is proceed in small volumes on microtiter plates
(microcytotoxicity tests – Terasaki plates)
HLA types described by the help of antibodies (or antibody
containing typing antisera) are called HLA serotypes
HLA serotypes:
HLA-D (HLA-DR, DQ, DP):
Dw „cellular HLA types” are described by
T cell alloreactions
Nomenclature for factors of the HLA system, 2010
Tissue Antigens 2010, 75, 291–455
(MLR – see it later)
Some features of the serotyping
• Serotyping identifies large “allelic groups” which contains similar
alleles
Genotyping could identify the exact allele, but the rapid serotyping narrows the possibilities
• w – workshop labelling: non characterized, ambiguous alleles
Their nomenclature can be changed in the future.
Most weakly immunogenic HLA-C and HLA-DP alleles have only “workshop” typing sera, so the
serotypes are also “w” labelled serotypes
• Numbers in the parentheses can refer to cross reactivity with other typing
serum, or can refer old “serogroups” which were discontinued after the exact
genotyping. e.g. A9  A23, A24 (see it later)
• MHC class II antigenes (HLA-DP, DQ, DR) can be found only on professional
APC. Peripheral blood B cells are used for it.
peripheral blood, Ficoll separation  PBMC (peripheral blood mononuclear cells)
PBMC, cheap nylon column  enriched B cells
(enriched B cells + EBV  transformed, immortal B cell lines)
Cellular reactions - Alloreactions
MHC compatibility can be examined by mixed lymphocyte reactions (MLR).
The presence of alloreactive T cells can be revealed by it.
Mononuclear cells of the donor and the recipient can be mixed. Alloreactive T cells will be activated,
and the proliferation can be examined by appropriate methods (e.g. 3H-thymidine incorporation)
• Two way MLR – both persons’ T lymphocytes can respond: It can be important
in the case of bone marrow transplantation to avoid graft versus host disease
• One way MLR – one person’s lymphocytes are prevented in the proliferation
(e.g. gamma-irradiation, Mitomycin C treatment)
The percentage of the alloreactive lymphocytes can be very high (1-10%)
The severe taboo mismatches can be screened with it
Allotypes of the HLA-D (MHC II) isotypes (DR, DQ, DP) can be examined by T
cells with known specific alloreactivity:
cellular typing
The cross reactions are rather frequent. “Dw” cellular types can be identified. (see the
table on the 2nd slide above) (w ! – workshop, ambiguous types !)
(The long term maintenance of the allospecific T cell lines are very difficult. The usage is very
limited)
Serotyping has limits:
• cross reactions are frequent (both the (polyclonal) antibody cross reactivity and the similar epitopes of
the MHC molecules should be considered)
• there are few typing sera against the HLA-C, HLA-DP isotypes (low immunogenity)
• small but important differences can’t be recognized by antibodies:
e.g. the T cell epitops can be located inside the proteins which can’t be accessed by the antibodies
Serology detects only the fragments of the full variability of HLA!
Molecular (genetic) typing
DNA sequencing has become wild-spread. Small sequence differences can
be shown very easily.
The DNA sequence differences can appear in the proteins:
“missense” variants – can influence both T and B cell recognition
“nonsense” variants – null alleles
Non exonic variants can influence the expression of the alleles which also
could influence the antigenity of the alleles
Molecular typing
• SBT (Sequence Based Typing)
DNA sequencing can be used to reveal the full sequence of an MHC
allele and able to identify small differences between sequences. Lots of
new alleles were identified this way.
MHC I
exons:
1
2
3
MHC II
4
5
67
leader,
signal
protein
domains
α-chain
8
n.t.
α1
α2
α3
1
2
4 56
leader,
signal
β1
tm c
α1 α2
β1 α1
α3
β2 α2
7
n.t.
α1
The exons encoding the most
polymorphic α1 and α2
domains are examined in the
routine typing (exon 2 and
exon 3 in this example)
3
β2
α2
tm c
β-chain
α-chain
The exons encoding the most
polymorphic domains of the
alpha and beta chains (α1
and
β1
domains)
are
examined in the routine typing
(exon 2 in this example)
Molecular (genetic) typing
• PCR-SSP (Sequence Specific Primer)
Used for typing alleles with known sequences
PCR examination with allele specific primer pairs recognizing allele specific HLA
sequences. The PCR multiplied product can be detected in the agarose gel at a specific
location.
HLA-A*
02:01 02:02
02:03
…
Molecular (genetic) typing
• PCR-SSOP (Sequence Specific Oligonucleotide Probes)
Labelled allele specific oligonucleotide sequences can hybridize to the DNA sample
of the examined person.
Used for typing alleles with known sequences
The most widely used HLA screening/typing method
•
•
•
•
The HLA region of the examined person is multiplied by PCR
The products are bound to a surface (immobilisation on membrane or microtiter plate)
The immobilised DNA can bind the allele specific labelled oligonucleotide probes
(hybridisation)
The label, as usual, can be radioactivity, fluorescence, or enzyme ( ELPHA)
An example for PCR-SSOP:
ELPHA (Enzyme Linked Probe Hybridization Assay)
(1st step)
- DNA isolation from PBMC
- Amplification of the MHC II genes’ 2nd exons and the MHC I genes’ 2nd
and 3rd exons by PCR. The primers are non allele specific and labelled
with biotin
DP
DQ
DR
Multiplied MHC “gene loci”
B
C
A
ELPHA (Enzyme Linked Probe Hybridization Assay)
(2nd step)
• The PCR amplificated DNA are distributed on microtiter plates covered with
streptavidine. The wells contain the allele sequence specific complementer
oligonucleotide probes (SSOP).
• The biotinylated DNA bind to the streptavidine.
• The complementer sequences hybridize. Only the bound sequences remain in
the wells after the washing.
allele specific oligo
biotinylated PCR product
streptavidine
The oligonucleotides are labelled/tagged. The tag is recognized by enzyme
conjugated antibodies which could generate colorisation
(The “tag” of the oligonucleotides should not be biotin, because the biotin is applied on the
amplificated DNA. They can use other tags: e.g. digoxigenin or FITC)
e.g. an HLA-A typing microtiter plate
A
B
C
HLA-A*02:76
D
E
HLA-A*24:34
F
G
H
1
2
3
4
5
6
7
8
9
10
11
12
Heterogeneity of the human MHC
http://hla.alleles.org/
DNA sequencing  The identified allele number is increasing year-by-year
MHC allele frequency groups:
common
- 0.1% (in at least 1500 examined person of a population)
well-documented
- described at least five times in unrelated individuals
rare
- reported 1-4 times from unrelated individuals
very rare
- reported one time
(common and well-documented alleles can be found in CWD HLA allele reports)
Nomenclature of the HLA system
(after 2010)
Huge number of different alleles  needs complex nomenclature
http://hla.alleles.org/
• The international HLA workshops regularely discuss te new allele and the
nomenclature.
• The new alleles are published here:
Tissue Antigens, Human Immunology, International Journal of Immunogenetics
• Large reviews and databases help to understand the nomenclature and the
“conversion” between the serotypic and the molecular nomenclature: Nomenclature
for factors of the HLA system, 2010, Tissue Antigens 2010, 75, 291–455
Connections between the genetic typing and the serotyping
The number after the asterisk is the “allele
group” which usually corresponds the serotype:
HLA-A*02  A2
*
e.g.:
Allele
name before
2010
serotype
There are exceptions:
Allele
name before
2010
serotype
The A*24 allele group usually corresponds the
A24(9) serotype
But the A*24:19 allele can be identified with only the
A9 specific typing serum
The A9 serotype (and former allelic group) was
separated to the new A*23 and A*24 allelic groups,
so there is no A*09 anymore
HYPERSENSITIVITY
REACTIONS
HYPERSENSITIVITY REACTIONS
Innocous materials can cause
hypersensitivity in certain
individuals
leading to unwanted inflammation
damaged cells and tissues
Non-proper reaction of the immune system to foreign substances
Mainly harmless substances – after second or multiple exposure
Different individuals respond differently to
allergens / hipersensitising agents
T cell response  MHC dependence
Atibody response generally needs also T cell activation
( MHC dependence)
MHC allelic heterogeneity in the population  Different
response from different individuals
AN OVERVIEW OF HYPERSENSITIVITY
REACTIONS
Type I.
Type II.
„immediate”
Type IV.
„late”
Antibody mediated
•
•
•
•
Type III.
T cell mediated
all start with sensitisation
autoimmune diseases can be connected with them
the effector mechanism are similar – so are the symptomps
there are few clear types – lots of overlapping properties between the different
types (Type I and Th2 Type IV can be connected; or Type IV neoepitops can
induce Type II like antibody response also)
TYPES OF ANTIBODY MEDIATED
HYPERSENSITIVITY REACTIONS
FcRIα)
TYPE I HYPERSENSITIVITY
REACTION
ALLERGY
(“Immediate” hypersensitivity reaction)
SENSITISATION PROCESS
Once sensitized (immunized), every following exposure
to the allergen elicits the allergic reactions.
ACUTE AND CHRONIC PHASE
OF THE ALLERGIC RESPONSE
Eosinophyls produces
toxic materials  tissue
damage
MAST CELL RESPONSE TO
SURFACE FcεR I CROSSLINKING
IgE
a
g b
I
I
TT
AA
MM
PLA2
foszfa tidil-kolin
LYSO-PC
arachidonsav
Fc eRI
I
T
A
M
MAPkináz
I
T
A
M
Lyn
II
TT
AA
MM
PIP2
Syk
endoplazmás
retikulum
2
Ca +
5-lipoxigenáz
PI-PLCg
DAG
IP3
foszfolipid
ciklooxigenáz
Ca 2+
PGD2
citokin gének
transzkripciója
LTC4
NFAT AP-1 NF-k B
PAF
szekretoros
granulum
2+
PKC Ca
Ca 2+
proteinek
(miozinkönnyűlánc)
foszforilációja
Ca 2+
szekréció
PGD2
LTC4
LTD4
LTE4
LATE MEDIATORS
(newly synthesized)
mediátorok
citokinek
IL-3, IL-4,
IL-5, IL-6
TNFa
EARLY MEDIATORS
(stored in granulums)
Biogenic amins – histamine
Enzymes – triptase, chymase, carboxypeptidase
early (stored) mediators
Examination of the mast cell function or the presence of antigen specific IgE
antibodies in laboratory animals:
Passive cutaneous anaphylaxis (PCA)
Antigen specific IgE can be administered intradermally, locally to an
animal (“local passive immunisation”)
Few days later intravenuos antigen and stain solution are
administered. (Evans-blue stain – can bind to albumin in the
circulation)
The small antigen can activate the sensitized mast cells locally
at the places of the local passive immunisation. The IgE carrier mast
cells can respond “immediately”.
The locally released histamine activates the small capillaries
and endothelial cells. The permeability is increased. The blue stain
enters in the tissues which can be observed by naked eye.
Mc-cpa−/− mast cells mount a normal passive cutaneous anaphylaxis reaction.
Thorsten B. Feyerabend et al. Mol. Cell. Biol. 2005;25:61996210
Skin test of allergy
Prick test
MAST CELL DEGRANULATION,
ALLERGIC REACTION IN THE SKIN OF
A SENSIBILIZED INDIVIDUAL
PRICK TEST
Allergic reactions composed of immediate
and late phase response some hours later
late phase mediators can
cause larger, long lasting
edema
ImmunoCAP
Specific IgE Blood Test
Allergen specific IgE can be shown
from the blood similarly
to the simple indirect ELISA method
Anti-IgE
Serum IgE
Allergen
Solid phase
Short/Common ragweed (Ambrosia artemisiifolia)
Short/Common ragweed (Ambrosia artemisiifolia)
Green leaf back
Mugwort (Artemisia vulgaris)
White leaf back
TYPE II HYPERSENSITIVITY
IgG type antibodies bound to
cell surface or tissue antigens
(usually)
• cells expressing the antigen become sensitive to complement
mediated lysis or to opsonized phagocytosis
• frustrated phagocytosis  tissue damage
• the antibody inhibits or stimulates target cell function
no tissue damage (e.g. M. gravis  receptor-blocking antibodies;
(Basedow-) Graves disease  TSH receptor stimulating antibodies)
MECHANISMS OF TYPE II HYPERSENSITIVITY REACTIONS
NK
Mf
Killing of target
cell by effectormacrophage or
NK-cell
IgG
ADCC
IgG
C'
complement
activation
Killing of target
cell by complementmediated lysis
Receptor-specific
autoantibody
interferes with
signal transduction
DEVELOPMENT OF DRUG
SENSITIVITY I.
neoepitope!
sensitisation
DEVELOPMENT OF DRUG SENSITIVITY II.
effector phase:
TYPE III HYPERSENSITIVITY
(Immune complex diseases)
Antibodies binding to soluble antigens
forming small circulating immune complexes
which are deposited in various tissues
Depends on:
• Size of immune complexes
• Antigen-antibody ratio (antigen excess in autoimmune diseases 
small complexes)
• Affinity of antibody
• Isotype of antibody
(see: precipitation/agglutination)
Large immuncomplexes are rapidly removed by phagocytic cells (FcR mediated)
THE PROCESS OF TISSUE DAMAGE CAUSED BY IMMUNE
COMPLEXES
Antigen
C'
Immune complex
Antibody
Complementa ctivation
(C3a , C5a )
PMN
Chemotaxis
C'
Endothelium Ba sophil
Ba sa l membra ne
gra nulocyte
Vessel wa ll
Thrombocytes
Deposition
Blood vessel
wall
permeability
Frustrated
phagocytosis
Vasoactive
a mines
Immune complexes activate the complement system,
neutrophils, basophils and thrombocytes
tissue penetrating anaphylatoxins (e.g. C5a) can activate mast cells also
SYMPTOMES CAUSED BY TYPE III HYPERSENSITIVITY REACTIONS
DEPEND ON THE SITE OF IMMUNECOMPLEX DEPOSITION
vasculitis symptoms in
serum sickness
ARTHUS-REACTION
• Localized Type III hypersensitivity
• Local vasculitis develops as a result of immune complex deposition
• Inhaled antigens (fungi, animal feces) may induce similar reaction in
the lung: ‘Farmers lung’ and ‘piegeon-breeder’s lung’
• IgG type antibody
DEPOSITION OF IMMUNE COMPLEXES IN THE SKIN OF
SLE PATIENTS (Immunofluorescence)
When immunofluorescence
staining with an antibody to
complement or immunoglobulin
is performed, a brightly
fluorescent signal staining the
dermal epidermal junction is
visible indicating immune
complex deposition.
Immunofluorescence staining pattern
with antibody to IgG staining immune
complexes at the dermal-epidermal
junction. If such a pattern is seen only in
skin involved by a rash, then the
diagnosis is probably DLE, but if this
pattern appears even in skin uninvolved
by a rash, then the diagnosis is SLE.
ANA
Anti-nuclear
antibody
This is the so-called "nucleolar pattern"
of staining in which the bright
fluorescence is seen within the nucleoli
of the Hep2 cells. This pattern is more
suggestive of progressive systemic
sclerosis.
This is the so-called "rim"
pattern that is more
characteristic of SLE.
This is the so-called "speckled" pattern
of staining which is more characteristic
of the presence of autoantibodies to
extractable nuclear antigens, particularly
ribonucleoprotein. This pattern is not
very specific, but may be seen with an
entity called "mixed connective tissue
disease" which is a mix between SLE,
scleroderma, and polymyositis, but
without serious renal or pulmonary
disease. The autoimmune diseases are
very hard to classify, even for the
experts.
TYPE IV HYPERSENSITIVITY
REACTION
T CELL MEDIATED PROCESS
(needs antigen presentation)
IV-es típusú túlérzékenységi reakció
(az effektor T sejtek alapján csoportosítva)
Type IV hypersensitivity is mediated by T cells
DELAYED-TYPE (TYPE IV)
sensitisation HYPERSENSITIVITY
DELAYED-TYPE (TYPE IV) HYPERSENSITIVITY
effector phase
DELAYED-TYPE HYPERSENSITIVITY (DTH)
examination: e.g. tuberculin skin test
TH1 from a previous
immunization (memory)
Tuberculin skin test
Introduction of Ag
Ag = antigen
Purified protein
derivate (PPD)
CHEMICAL MEDIATORS OF DTH
cytokine detection methods: ELISA…..etc
DTH as a result of a contact-sensitizing agent*
CONTACT DERMATITIS
*a contact-sensitizing agent is usually a small molecule that penetrates the skin
then binds to self-proteins, making them “look” foreign (T cell neoepitope)
Plant containing contact sensitizing agent
Poison ivy
Anacardiaceae (family), Toxicodendron (genus)
Toxicodendron radicans or Rhus toxicodendron
(North America)
Kontakt szenzitizálás hatására
lipids with catechol ring
(Urushiols):
R = (CH2)14CH3
R = (CH2)7CH=CH(CH2)5CH3
R = (CH2)7CH=CHCH2CH=CH(CH2)2CH3
R = (CH2)7CH=CHCH2CH=CHCH=CHCH3
R = (CH2)7CH=CHCH2CH=CHCH2CH=CH2
etc.
(neoepitopes)
CELIAC DISEASE