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FLOW CYTOMETRY
An immunofluorescent method that mutually
complements the fluorescent microscopy
Detection and analysis of different cells or particles travelling
at high velocity in flow
Detects fluorescence intensity and scattered light of the
labeled cells
Can investigate enormous number of cells in a short period
of time
ADVANTAGES OF FLOW CYTOMETRY
Most cells in the immune system can be found in free or loosely
adherent form. They can be easily suspensed and labeled by
fluorescent antigen specific antibodies, and then they can be examined
cell by cell
The cells’ light scatter and immunofluorescent properties can be
analyzed statistically (e.g. percentages of different cell populations)
Rare cell populations can be identified and examined (e.g. antigen
specific lymphocytes)
The method provide qualitative and quantitative data – it can detect
the presence of different antigens in the cell, and the expression levels
of these antigens. Changes in the expression of certain molecules can
be followed after different treatment of the specimen. (e.g. cell
activation, disease progression)
Benchtop flow cytometer
Sorter - flow cytometer
(FACS station)
LIGHT SCATTER AND FLUORESCENCE
Forward angle light
scatter sensor
(FSC, FALS)
Laser
Can be loosely
considered as a
representation of the
particle size
Side light scatter (SSC) and fluorescence detectors
SSC represents the granularity of the cells
Multicolor staining can be used to identify cell sub-populations
(autofluorescence – presence of piridins and flavins)
IMMUNOPHENOTYPING BY FACS
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(e.g. monitoring AIDS progression)
Labeling:
Th
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
NK
Tc
Lymphocytes in the peripheral blood sample
Fluorescent microscopy
B
high velocity flow stream
detecting CD4-FITC
labeled (TH) cell
(in cuvette or stream in air)
signal processing
unit
CD8
PE
detector
screen
increasing
light intensity
a dot representing a
CD4+ CD8- cell
CD4
FITC
microscopy:
detecting the PE labeled cell
(CD8-PE)
CD8
PE
detector
signal processing
unit
increasing
light intensity
CD4
FITC
detecting the unlabeled cell
(e.g. B cell) by autofluorescence
CD8
PE
detector
Signal processing
unit
increasing
light intensity
microscopy:
dim (autofluorescent) cell
CD4
FITC
CD8
PE
18%
44%
0%
quadrant
statistics
CD4 38%
FITC
GRAPHICAL REPRESENTATIONS 1.
dot-plot
contourplot
densityplot
GRAPHICAL REPRESENTATIONS 2.
Histogramm
Numeral intensity
values:
~7
~ 1300
homogenous cell
population is normally
distributed (Gaussian)
Different cell types - characteristic light scattering
granulocytes
side light scattering (SSC)
(e.g. granulated)
monocytes
lymphocytes
forward light
scattering (FSC)
(„size”)
EXAMINATION OF PERIPHERAL BLOOD BY HAEMATOLOGY AUTOMATS
Measured parameters:
peroxydase staining (the presence of myeloperoxydase, x – axis)
light scatter (high on large granular cells, y – axis)
1 (Noise)
2 Nucleated Red Blood Cells
3 Platelet Clumps
4 Lymphocytes and Basophils
5 Large Unstained Cells (LUC)
6 Monocytes
7 Neutrophils
8 Eosinophils
Only the major cell types can be identified
CHARACTERIZATION OF IMMUNE CELLS
USING CELL SURFACE MARKERS
Cell types, differentiation stages can be identified using a combination of
cell surface markers.
Used in diagnostics:
- ratio of different cell types
- altered expression of cell surface markers
Examples:
- Inflammatory processes – increased neutrophil numbers
- HIV progression – decrease of CD4+ T cell count
CD4+ : CD8+ = 1.6
Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
- increase of CD5+ B cells – typical for some B cell leukemias
CD antigen
cell type
function
ligand
CD3
T cells
TCR signaling
-
CD4
helper T cells, (monocytes, pDC)
T cell co-receptor, (HIV
receptor)
MHC- II, HIV
CD5
T cells, (B cell subset: B1)
adhesion, activation signals
CD72
CD8
cytotoxic T cells, (NK,  T cells)
T cell co-receptor
MHC I
CD14
monocytes, macrophages,
some granulocytes
LPS binding
LPS, LBP
CD19
B cells
part of CR2, B cell coreceptor
C3d, C3b
CD28
T cells
co-stimulatory signals to T
cells
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cell
adhesion
CD62L
(L-selectin)
CD56
NK cell, (T and B cell subset)
homoadhesion (N-CAM
isoform)
APC: DC, B, monocyte,
macrophage
co-stimulatory signals
CD80, CD86
(B7-1, -2)
CD28, CD152
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 cytokines by flow
cytometry
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
first with a cell type specific antibody
(e.g. CD4)
cytokines
The result:
You can determine which cell type has produced the
cytokines!
Sensitive, relatively easy method, rare cell populations
can be readily studied. Multiple colours are available
for detection of more cytokines or cell surface markers
The cell cycle can be examined by a fluorescent dye
G2
G0
M
that intercalates stoechiometrically
into double stranded DNA
(e.g. propidium iodide, PI)
DNA analysis
G1
G0 G1
s
cell number
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 by flow cytometry:
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:
8 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
MHC multimer 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
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.
T cell receptors
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 (from the ’90ies)
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 jirovecii. 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
jirovecii, Helen was suspected to have severe combined immunodeficiency.
What kind of laboratory test should be performed to
confirm 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
3H-thymidine 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 when she was 2 months old.
However, in further tests, her T cells failed to respond to tetanus toxoid 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 (normal range: 10-33%),
10% lymphocytes (n.r: 20-40%),
6% monocytes (n.r: 2-10%),
and
2% eosinophils (n.r: 1-6%).
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 lower than the normal range (5001700/μl), and 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:
1400-4300).
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 low level of immunoglobulins in her blood?
The polyclonal expansion of B lymphocytes and their maturation to
immunoglobulin-secreting plasma cells require cytokines from CD4 T helper 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 CD4 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.