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9. seminar/practice
Methods to measure functional of the
immunocompetent cells
blast transformation (LPS and ConA activation),
polyclonal B and T lymphocyte activation,
ELISPOT
Measuring the functional activity of T
and B lymphocytes
Topics:
Polyclonal activation of T and B cells via non-antigen-specific
stimulation
lectin-induced activation
α-IgM, α-CD3 or α-TCR antibody
allogeneic T cell activation
(examination of the immediate-early activation events)
Characterization of responses by activated T and B cells
activation markers
proliferative response: 3H-thymidine incorporation
CFSE fluorescence decrease
cell cycle events
Antibody or cytokine production (ELISA, CBA)
Determination of the number of activated T and B cells after the
administration of the antigen
ELISPOT,
Intracellular cytokine staining
Pentamer (or tetramer) technics
(review)
Phases of the humoral immune response
(review)
Phases of T cell response
(review)
BCR signaling
(review)
TCR signaling
Immunodeficiencies mainly characterized by different
functional immunoassays
Lymphocyte activation by specific antigen is hardly detected,
because of the low number of the antigen specific cells
Lymphocyte function can be investigated by polyclonal T/B-lymphocyte
activator materials
Polyclonal activation of lymphocites by
LPS, lectins, PMA/ionomycin
B cell (mouse)
T cell
T cell
TLR4
(PMA activates protein kinase C)
BCR- or TCR-specific antibodies may also activate the lymphocytes
Polyclonal B cell activators
Activator
T cell dependency
Ig secretion
Human B cells
PWM (pokeweed mitogen)
no
yes
SpA (superantigen, staphylococcus protein A)
no
yes
EBV (transforming effect)
yes
yes
Anti-Ig
yes
In the presence of cytokines
Mouse B cells
LPS
no
yes
PWM
yes
yes
PPD (purified protein derivate, mycobacterium) no
yes
Anti-Ig
In the presence of cytokines
no
Polyclonal T cell activators
Phytohaemagglutinin (PHA)
lectin
Canavalia ensiformis
Concanavalin A (ConA)
lectin
Phaseolus vulgaris
anti-CD3
Monoclonal antibody
Pokeweed (PWM)
(Phytolacca americana) – formerly
used for colouring red wine
(toxic: triterpene saponin)
Chenopodiales
Phytolaccaceae
Phytohaemagglutinin (PHA)  Canavalia ensiformis – Jack-bean, Sword bean
Concanavalin A (ConA)  Phaseolus vulgaris – bean
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
Western blot
It can detect the presence or even phosphorylation
state of specific proteins
The cells’ activation stage can be „frozen” at different times, so
the events of the activation can be monitored in parallel
samples.
at least 105-106 cells required
Investigation of the presence or absence of
Bruton’s tyrosine kinase (BTK) by Western blot
X-linked agammaglobulinemia. XLA patients do not generate mature B cells,
which manifests as an almost complete lack of antibodies in their bloodstream.
Investigation of the presence or absence of
Bruton’s tyrosine kinase (BTK) by flow cytometry
Futatani T et al. Blood 1998;91:595-602
Detection of intracellular (cytoplasmic) Ca2+ concentration
An inrease in cytoplasmic Ca2+
levels can be detected by
fluorescent indicator dyes.
/Fluo-3 or Indo-1/
for example – ic Ca2+ signal in a single cell
antigen presentation by B cell to T cell
(click)
Investigation of gene activation
Activation of T cells can be monitored by the detection
of the transcribed mRNA of the activated genes.
e.g. activation of cytokine genes
method: RT-PCR, QRT-PCR
cells  RNA isolation
RNA  (reverse transcriptase)  cDNA
cDNA  (PCR)  determination of the length and quantity
RT-PCR: agarose gel (densitometry)
QRT-PCR: fluorescent method
(TaqMan probe (FRET) or dsNA intercalating fluorochrome  SYBR green)
Intracellular cytokine detection by
immunofluorescence
cytokine specific antibody with fluorescent labelling
- the cell membrane should be permeabilized (detergent)
- the cells should be fixed previously avoiding the
decomposition of the cells (e.g. aldehyde fixation)
- optionally the cells could be
labelled by some cell type specific
antibody in the beginning (e.g. CD4)
cytokines
The result:
You can determine which cell type has produced
the cytokines!
The sensitivity could reach that of the Western blot.
(e.g. with chilled CCD camera mounted microscope – but you need only
one cell for detection)
ELISPOT
Enzyme Linked Immuno-Spot
the principles are similar to ELISA
capable to determine the number of cells that produce Ig,
cytokines, chemokines, granzymes and other soluble
effector molecules
the sensitivity allows the determination 1 activated cell
among 300 000 other, so it can reveal activated effector
cells not only after policlonal-, but after antigen specific
activation
the first steps should be done in aseptic conditions
ELISPOT
The process
- coating with antigen specific capture antibodies
- blocking
- administration of the cells (activation, incubation)
- washing
- administration of biotin conjugated
antigen specific secondary antibody
- avidin-enzyme conjugate
upper view of a well on
an ELISPOT plate with
- administration of the chromogenic
the generated spots
substrate (AEC 3-amino-9-ethylcarbazol)
A spot showing
the place of the
cytokine
producing cell
It can be evaluated by microscopy (slow, manual process)
or you can use “ELISPOT plate reader” (fast + standardizable spot
number and size determination)
The size of the cycling cells are increased –
called blast transformation
Cell-cycle
Possibility of the examination
Stimuli
(e.g. antigen)
resting lymphocyte
(G0)
effector cell
- transcription (RT-PCR)
- protein synthesis
memory cell
(Immunoassay)
changes in the RNA- and protein
synthesis, in the cell membrane
and in the transports
cell division
change in the
number of the cells
DNA-synthesis
(MTT, CFSE)
DNA quantification
(fluorescent DNS intercalating agents,
3H-thymidine)
The cell cycle can be examined by fluorescent dye
G2
G0
M
that intercalates stoechiometrically
into the double stranded DNA
(e.g. propidium iodide, PI)
DNA analysis
G1
cell number
s
G0G1
G2 M
s
0
200
400
600
800
1000
4N
2N
DNA content
Distribution of a normal cycling cell-population by
DNA content (flow cytometry)
Methods for determinating the
B/T cell proliferation
3H-labeled
thymidine incorporation – measures the increasing DNA
content by β decomposition, and does not answer the numbers of cell
division, and the dividing cell number
thymidine-analog bromodeoxyuridin (BrdU) can be administered to
experimental animals, or cell cultures, and the proliferating cells can be
detected by labelling with BrdU specific antibody (microscopy, FACS)
Carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescent
stain can be used to tracking the cell divisions:
Tracking the cell divisions
„Cell tracer” dye enter the cell, and trapped there.
The apolar CFSE can bind covalently to the cellular
proteins. Later the stain can only be diluted by the cell
divisions: distributed equally between the two daughter
cells – the fluorescence intensity decreases to the half also.
cell divisions:
7 6 5 4 3 2 1 0
T cell antigen specificity
Identifying the antigen specific T cells
The efficiency of an immunization can
be evaluated by the increase of the
antigen specific cell number
antigen
specific
T cell
T cell clones with the
same T cell receptor
immunization
If you can identify the specificity of the T cell receptors then you can
monitor the increase of the antigen specific T cells’ number
Labelled MHC-peptide complex can be used to
identify the matching (specific) T cell receptor
..but the MHC binds the TCR with low affinity
MHC
T cell receptors
T cell
The interaction
between one MHC
molecule and one TCR
is not strong enough
for labelling
The multimerized MHC-peptide complex can have enough avidity
Pentamer (or tetramer) technics
One part of the pentamer
peptide
MHC
molecule
self assembling
coiled-coil-domain
fluorescent label
The pentamer
Binding of the MHC pentamer to the T-cell
MHC pentamer
T cell receptors
The MHC-peptide oligomer can
bind the specific T-cell receptors
with high avidity
The number of the antigen-specific T
cells can be evaluated by MHC
multimers. So the efficiency of an
immunization or a therapy can be
estimated.
peptide specific
T cell
Click here to watch
the animation
EBV BZLF-1 (RAKFKQLL/
HLA-B*0801) specific T cells
(90-95% of the human population are carrier)
Tetramer (pentamer) tests
The number of microbe specific T cells
can be increased in the body because
of the persistent (e.g. herpesviruses) or
repeated infections
CMV specific T cells in healthy
HLA-A2 donor
Influenza epitope (GILGFVFTL/
HLA-A0201) specific T cells in a
healthy donor
allele
sequence
Tumour (associated) epitope
A*0201
GVLVGVALI
Carcinogenic Embryonic Antigen (CEA) 694-702
A*0201
LLGRNSFEV
p53 261-269
A*0201
LLLLTVLTV
MUC-1 12-20
MHC-peptid pentamers for detecting
antigen specific T cells
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
allele
sequence
A*0201
VISNDVCAQV
Prostate Specific Antigen-1 (PSA-1) 154-163
A*0201
CLGGLLTMV
EBV LMP-2 426-434
A*0201
VLQELNVTV
Leukocyte Proteinase-3 (Wegener's autoantigen) 169-177
A*0201
GLCTLVAML
EBV BMLF-1 259-267
A*0201
VLYRYGSFSV
gp100 (pmel17) 476-485
A*1101
IVTDFSVIK
EBV EBNA-4 416-424
A*0201
YLEPGPVTA
gp100 (pmel17) 280-288
A*2402
TYGPVFMCL
EBV LMP-2 419-427
A*0201
YLSGANLNL
Carcinogenic Embryonic Antigen (CEA) 571-579
B*0702
RPPIFIRRL
EBV EBNA-3A 247-255
A*0201
KVLEYVIKV
MAGEA1 278-286
B*0801
FLRGRAYGL
EBV EBNA-3A 193-201
A*0201
KVAELVHFL
MAGEA3 112-120
B*0801
RAKFKQLL
EBV BZLF-1 190-197
A*0201
KTWGQYWQV
gp100 (pmel17) 154-162
B*3501
HPVGEADYFEY
EBV EBNA-1 407-417
A*0201
HLSTAFARV
G250 (renal cell carcinoma) 217-225
A*0201
ILAKFLHWL
Telomerase 540-548
allele
sequence
Influenza A epitope
A*0201
ILHNGAYSL
HER-2/neu 435-443
A*0101
CTELKLSDY
Influenza A (PR8) NP 44-52
A*0201
IMDQVPFSV
gp100 (pmel17) 209-217
A*0201
GILGFVFTL
Influenza A MP 58-66
A*0201
KIFGSLAFL
HER-2/neu 348-356
A*0301
ILRGSVAHK
Influenza A (PR8) NP 265-274
A*0201
LMLGEFLKL
Survivin 96-104
A*0201
ALQPGTALL
Prostate Stem Cell Antigen (PSCA) 14-22
allele
sequence
A*0201
CMTWNQMNL
Wilm's Tumour (WT1) 235-243
A*0201
ILKEPVHGV
HIV-1 RT 476-484
A*0201
ELAGIGILTV
MelanA / MART 26-35
A*0201
KLTPLCVTL
HIV-1 env gp120 90-98
A*0201
FLTPKKLQCV
Prostate Specific Antigen-1 (PSA-1) 141-150
A*0201
SLYNTVATL
HIV-1 gag p17 76-84
A*0201
GLYDGMEHL
MAGEA-10 254-262
A*0201
TLNAWVKVV
HIV-1 gag p24 19-27
A*0301
KQSSKALQR
bcr-abl 210 kD fusion protein 21-29
A*0301
QVPLRPMTYK
HIV-1 nef 73-82
A*0301
ATGFKQSSK
bcr-abl 210 kD fusion protein 259-269
A*0301
RLRPGGKKK
HIV-1 gag p17 19-27
A*0301
ALLAVGATK
gp100 (pmel17) 17-25
A*2402
RYLKDQQLL
HIV-1 gag gp41 67-75
A*2402
VYGFVRACL
Telomerase reverse transcriptase (hTRT) 461-469
B*0702
IPRRIRQGL
HIV-1 env gp120 848-856
A*2402
TYLPTNASL
HER-2/neu 63-71
B*0702
TPGPGVRYPL
HIV-1 nef 128-137
A*2402
TYACFVSNL
Carcinogenic Embryonic Antigen (CEA) 652-660
B*0801
FLKEKGGL
HIV-1 nef 90-97
A*2402
TFPDLESEF
MAGEA3 97-105
B*0801
GEIYKRWII
HIV-1 gag p24 261-269
A*2402
EYLQLVFGI
MAGEA2 156-164
B*2705
KRWIILGLNK
HIV-1 gag p24 265-274
A*2402
CMTWNQMNL
Wilm's Tumour (WT1) 235-243
H-2Kd
AMQMLKETI
HIV-1 gag p24 199-207
A*2402
AFLPWHRLF
Tyrosinase 188-196
B*0801
GFKQSSKAL
bcr-abl 210 kD fusion protein 19-27
EBV epitope
HIV epitope
Case study
Helen Burns was the second child born to her parents. She thrived until 6
months of age when she developed pneumonia in both lungs, accompanied by a
severe cough and fever. Blood and sputum cultures for bacteria were negative
but a tracheal aspirate revealed the presence of abundant Pneumocystis carinii.
She was treated successfully with the anti-Pneumocystis drug pentamidine and
seemed to recover fully.
As her pneumonia was caused by the opportunistic pathogen Pneumocystis
carinii, Helen was suspected to have severe combined immunodeficiency.
What kind of laboratory test should be
performed to make or rule out the diagnosis
of severe combined immunodeficiency?
A blood sample was taken and her peripheral blood mononuclear cells were
stimulated with phytohemagglutinin (PHA) to test for T cells function by 3Hthymidine incorporation into DNA.
A normal T-cell proliferative response was obtained, with her T cells
incorporating 114,050 counts/min of 3H-thymidine (normal control 75,000
counts/min).
Helen had received routine immunizations with orally administrated polio
vaccine and DPT (diphtheria, pertussis, and tetanus) vaccine at 2 months old.
However, in further tests, her T cells failed to respond to tetanus toxin in vitro,
although they responded normally in the 3H-thymidine incorporation assay
when stimulated with allogeneic B cells (6730 counts/min incorporated
compared with 783 counts/min for unstimulated cells).
When it was found that Helen's T cells could not respond to a specific antigenic
stimulus, her serum immunoglobulins were measured and found to be very low.
IgG levels:
IgA levels:
IgM levels:
96 mg/dl (normal: 600-1400 g/dl)
6 mg/dl (normal: 60-380 mg/dl)
30 mg/dl (normal: 40-345 mg/dl)
Helen's white blood cell count was elevated at 20,000 cells/μl (normal range
4000-7000/μl).
Of these, 82% were neutrophils,
10% lymphocytes,
6% monocytes,
and
2% eosinophils.
The calculated number of 2000 lymphocytes/μl is low for her age
(normal >3000 μl-1).
Of her lymphocytes,
7% were B cells (CD20+) (normal 10-12%)
57% reacted with antibody to the T cell marker CD3.
At 388 cells/μl her number of CD8+ T cells was within the normal range, but the
number of CD4+ T cells (288/μl) was much lower than the normal (her CD4+ T-cell
count would be expected to be twice her CD8+ T-cell count).
The presence of substantial numbers of T cells, and thus a normal response to
PHA, ruled out a diagnosis of sever combined immunodeficiency.
Helen's paediatrician referred her to the Children's Hospital for consideration for
a bone marrow transplant, despite the lack of diagnosis. When an attempt was
made to HLA type Helen, her parents and her healthy 4-year-old brother, a DR
type count not be obtained from Helen's white blood cells. A long-term culture
of her B cells was made by transforming them with Epstein-Barr virus and the
transformed B cells were then examined for expression of MHC class I and
class II molecules with fluorescent-tagged antibodies. It was found that her B
cells did not express HLA-DQ or HLA-DR molecules and a diagnosis of
MHC class II deficiency was established.
Detection of MHC class II
molecules by fluorescent antibody.
Helen’s transformed B-cell line was
examined by using a fluorescent antibody to
HLA-DQ and –DR. Helen (left panels)
expressed approximately 1℅ of the amount
of MHC class II molecules compared with a
transformed B-cell line from a normal control
(right panels).
Cells from a patient with
class II histocompatibility deficiency
Immunofluorescence of normal
EBV-transformed cells
Transformed B cells
Transformed B cells
Transformed B cells
Transformed B cells
As her brother did not have the same HLA type as Helen, it was decided to use
her mother as a bone marrow donor. The maternal bone marrow was depleted of
T cells to diminish the chance of graft-versus-host disease developing and was
administered to Helen by transfusion. The graft was successful and immune
function was restored.
Discussion and questions
1. Why did Helen lack CD4 T cells in her blood?
The maturation of CD4 T cells in the thymus depends on the interaction of thymocytes
with MHC class II molecules on thymic epithelial cells. When the MHC class II genes
are deleted genetically in mice, the mice also exhibit a deficiency of CD4 T
lymphocytes.
2. Why did Helen have a low level of immunoglobulins in her blood?
The polyclonal expansion of B lymphocytes and their maturation to immunoglobulinsecreting plasma cells requires helper cytokines from CD4 T cells, such as IL-4.
Helen’s hypogammaglobulinemia is thus a consequence of her deficiency of CD4 T
lymphocytes.
3. In SCID , lymphocytes fail to respond to mitogenic stimuli. Although Helen
was first thought to have SCID, this diagnosis was eliminated by her normal
response to PHA and an allogenic stimulus. How do you explain these findings?
Helens’ T cells, although decreased in number, are normal and are not affected by the
defect. They are capable of normal responses to nonspecific mitogens and to an
allogenic stimulus in which the antigen is presented by the MHC molecules on the
surface of the (nondefective) allogeneic cells and thus does not require to be
processed and presented by the defective cells. However, the failure of her
lymphocytes to respond to tetanus toxin in vitro resulted from the fact that, in this
situation, there were no cells that could present antigen on MHC class II molecules to
the CD 4 T cells.
4. If a skin graft were to be placed on Helen’s forearm do you think she would
reject the graft?
Yes. Helen’s T cells would be capable of recognizing the foreign MHC molecules on
the grafted skin cells and would reject the graft.