Download B cells

Joseph Cannon, PhD
Clinical Laboratory Sciences Program
Augusta University
Lecture overview
Antibody synthesis
(B cells)
diversity
specificity
Antibody structure
characteristics of monomers
isotypes
binding sites
Distribution
Laboratory reagent
body compartments
epithelial transport
fetal environment
diagnostic in vitro assays
In vivo imaging
therapy
Function
interaction with antigen
microbial evasion
interactions with host cells
(and other host defense molecules)
Antibody synthesis
• B cells
• diversity
• specificity
The source of antibodies
B cells express CD19
4-8% of circulating leukocytes
Responsible for humoral immunity ≡ antibody-mediated
differentiate into memory cells or plasma cells
CD19
B
CD24
CD20
the B cell receptor is a monomeric form of IgM
plasma cells secrete antibodies
B cell development
CD19
• CD19 is expressed early (Pro-B cell)
CD19
• B cell receptors (monomeric IgM)
develop in bone marrow phase and
are present on the surface of
immature cells
CD19
• IgD is expressed on B cells
in later stage of development “mature” B cells
• “virgin” means that the cell has not
yet encountered the antigen that will
stimulate it
• class switching and somatic mutation
occur after stimulation with antigen
CD19
• remember that memory cells are also
generated after antigen stimulation
CD19
Figure: Rittenhouse-Olsen & De Nardin
Antigen-independent Diversity
(random recombination of gene segments to create heavy chains)
V1 V2 V3 Vn
D1 D2 D3 Dn
J1 J2 J3 Jn
ǁ
ǁ
ǁ
excised
V1 V2 V3 Vn
Cm Cd Cg3 Cg1 Cg2 Cg4 Ce Ca
D/J joining
D1 D2J3 Jn
ǁ
DNA
Cm Cd Cg3 Cg1 Cg2 Cg4 Ce Ca
ǁ
excised
DNA
V/D/J joining
V1 V2D2J3 Jn
Cm Cd Cg3 Cg1 Cg2 Cg4 Ce Ca
ǁ
DNA
Transcription and splicing
V2D2J3Cm
mRNA
Translation
A similar process (on different
chromosomes) creates the light
chains (but no D segments). These
chromosomes have a segment that
codes for k or l constant region.
V2D2J3Cm
IgM heavy chain
Cytokines from TH cells signal
for excision of Cμ gene and
recombination of VDJ region
with a different constant gene
(e.g., Cg, resulting in IgG)
Clonal expansion
Figure: Germann & Stanfield
clonal selection
Diversity: Millions of
lymphocytes exist, each
one recognizes only a
single epitope on a single
antigen.
proliferation
and differentiation
of clones
Plasma cells
(effectors)
Memory
B cells
secrete
antibodies
All clones share same antigen specificity
bind antigen
Memory and effector T cells are
generated the same way.
Polyclonal antibodies
• Most antigens have many epitopes
• Therefore not one, but many B cells are
activated
• When an animal or human is vaccinated,
antibodies against each of the epitopes
are produced and circulate
• Therefore, serum contains polyclonal
antibodies
Different
epitopes
Antigen
B cell
Immunological Memory
Second
exposure
to the same
antigen
Antibody structure
• characteristics of monomers
• isotypes
• binding sites
Immunoglobulin classification
Isotype
Isotype refers to major
classes of antibodies
(IgA, IgG, IgM, IgD, IgE),
based on heavy chain
constant regions (a, g, m,
d, e). Isotype is relatively
constant within each
species.
Allotype
Allotype refers to small
differences among
individuals in the constant
regions of light and heavy
chains (inherited).
Idiotype
Idiotype refers to the
specific binding behavior
conferred by hypervariable
region (antigen binding site).
Two types of light chains: kappa (k) and lambda (λ) exist in 2:1 ratio
(no known functional differences)
Isotypes
IgG
IgM
IgA
IgD
IgE
U
U
# of HC domains:
4
5
4
4
5
‘meric structure:
mono-
penta-
di-
mono-
mono-
# antigen binding
sites
2
10
4
2
2
Monomer characteristics
Heavy chain (Fc) is constant
for each Immunoglobulin class
(isotype) within each species.
Enzymatically-produced fragments
The two antigen binding sites
remain connected
F(ab’)2
Fc’
Pepsin breaks heavy chains below the
disulfide bonds that connect them.
F(ab’)2: minimum structure
capable of agglutination
Antigen binding sites are
separate
Fab
Fab
Fc
Papain breaks heavy chains above the
disulfide bonds that connect them.
Fab: minimum steric hindrance
Fc is the key domain for:
• Distribution in body (neonatal FcR)
• Binding to specific cells (Fcg -vs- Fce)
• Classical complement activation (C1q)
• Anchor points for J chain
• Recognizing/measuring patient antibody
• Microbial evasion
IgG
• ~80% of total Ig in serum
• Passes into interstitial fluid
• Biological functions:
o agglutination
o neutralization
o activates (“fixes”) complement
o opsonization (Fcg receptors on phagocytic cells)
o promotes antibody-dependent cell-mediated cytotoxicity (ADCC)
• Passes through placenta, confers infant protection for ~6 months
IgA
U
• ~10% of total serum Ig (monomer: has little apparent function)
• Dimer is secreted onto mucosal surfaces, preventing pathogen entry
• 2 Fc’s joined by J chain
• Secretory piece added in passage through epithelial cell
• Agglutinates, neutralizes, opsonizes
• Transferred to infants through breast milk and colostrum, protects
against enteric pathogens
IgM
• Largest mass: 900 kDa
(macroglobulin)
U
• Pentamer
• ~10% of total serum Ig
• 10 antigen binding sites
(each with low affinity, but high total avidity)
• First Ig produced, no somatic mutation
• Can be secreted (J chain binds to poly-Ig receptor)
• Surface receptor on B cells (as monomer)
• Best preciptator, agglutinator and complement fixer.
Also opsonizes and neutralizes.
Flexibility of IgM
Figure: Kuby et al.
Efficient at agglutination
IgE
• Very little in serum (0.02% of Ig)
• Has extra domain in the heavy chains
(compared to IgG)
• Made by plasma cells near interface
with external the environment
• Mast cells, basophils, eosinophils, and Langerhans cells* have Fce receptors
• Induces degranulation by mast cells (release of histamine, heparin &
chemoattractants)
• Mediates allergic responses
• Mediates protective responses against pathogens that penetrate mucosal
barriers (especially parasites)
• Does not agglutinate, opsonize, or fix complement
*specialized dendritic cells in the skin
IgD
• 0.2% of serum Ig
• antigen receptor on B cells
appearing in late stage of
development (“mature”)
• Responds to T cell signals
for class switching
(eg. IgM to IgG)
• does not agglutinate,
opsonize, or fix complement
Antibody distribution
• body compartments
• epithelial transport
• fetal environment
Distribution of Immunoglobulins
• Heart represents bloodstream
in this figure.
• In addition to bloodstream, IgM
also concentrated in pleural &
peritoneal spaces (also some
secretion)
from: Janeway’s Immunobiology 8, Garland Science, 2011
Transepithelial passage of IgA
U
Plasma cell
IgA poly-Ig
receptor
Epithelial cell
U
endocytosis
U
exocytosis
U
transcytosis
IgA + secretory
component
Transplancental passage of IgG
same mechanism
for transendothelial
IgG passage
plasma
neonatal Fc
receptor
endothelial
cell
extracellular
fluid
Antibody function
• interaction with antigen
• microbial evasion
• interactions with host cells
and molecules
Antibody function #1: Opsonization
Coating antigen with antibody enhances
phagocytosis
Phagocyte
Phagocytosis of Opsonized Pathogen
Fc receptor
Pathogens coated with antibodies, CRP, or C3b
Phagocytosis of Opsonized Pathogen
“Zipper effect”
Phagocytosis of Opsonized Pathogen
Pathogen evasion mechanism
protein A
Staphylococcus
aureus
Protein A binds Fc region of antibodies,
therefore Fc receptors on phagocytic cells cannot bind
Antibody function #2: Agglutination
The basis for many non-labeled serological assays
Unlabeled Immunoassays
• Can actually see the formation of immune complexes, either by their
ability to scatter light (precipitation) or the actual complexes
themselves (agglutination).
• Unamplified reaction, so not very sensitive (~20 μg/ml for
precipitation).
• Optical equipment can improve sensitivity (turbidimetry and
nephelometry) to ~1 μg/ml (for nephelometry)
• Each antigen and antibody must have at least 2 binding sites in order
for complexes to form.
Precipitation
Agglutination
Agglutination of red blood cells
U
Zeta potential (surface charge):
RBCs typically separated by 25 nm
(IgM diameter = 35 nm)
IgM agglutinates best at 4-27°C
IgG is best at 37°C
Ag binding sites must equal Ab binding sites
Zone of equivalence
Antigen/antibody complexes
Prozone
Antigen concentration
Postzone
Antibody function #3: Neutralization
Antistreptolysin O test
Anti-strep antibodies are detected in vitro by the ability of a
patient’s serum sample to neutralize the bacterial exotoxin
Antibody function #4: Complement activation
(by the classical pathway)
(C1)
Antibody function #5: ADCC
Antibody-dependent cell-mediated cytotoxicity
Eosinophil
epitopes
perforin &
lytic enzymes
IgE
Fce
Appropriate, defense response
Antibody as a
laboratory reagent
• production
• application
• problems
Polyclonal antibody production
Different
epitopes
Antigen
•
•
•
•
Immunize animal
Take blood
Whole serum
(NH4)2SO4 precipitate
• Affinity purification (protein A)
• Antigen-affinity purification
Proteins
Lipids
CHO
Large
proteins
Single
isotype
Ig
Single
antigen
Ig
X
X
X
X
many
idiotypes
Monoclonal Antibodies
Mouse is injected
with antigen
Many antibody-producing
B cells in spleen
Spleen cells mixed
with myeloma cells,
some fuse together
(hybridize)
In “HAT” media,
only hybrid cells
survive
Single hybrid cells
in separate wells
Single hybrid cells
proliferate, antibody
secreted is screened
Desired hybridoma is cultured, large
amounts of monoclonal antibody produced
Hybridomas
Labeled Assays
Competitive Immunoassay
• Incubate samples with
immobilized antibody (or antigen)
sample A
sample B
• Add labeled analyte to sample.
• Unlabeled and labeled analytes
compete for binding sites.
• Wash away unbound material
(then add substrate).
• Measure signal generated by label
(radioactivity, color, light, or polarity).
Signal
• Signal measured is inversely
related to analyte concentration.
A
B
Analyte
Concentration
Noncompetitive Immunoassay
sample A
sample B
• Incubate samples with immobilized
capture antibody (or antigen)
• Wash away unbound material
• Add labeled detection antibody
• Wash away unbound material
(then add substrate)
• Signal measured is directly
proportional to analyte conc.
• Indirect: label not involved in
first antigen-antibody reaction
• “Sandwich” assay
Signal
• Measure signal generated by
bound, labeled antibody
B
A
Analyte
Concentration
Antibody
capture
Interfering Substances
Rheumatoid factor
Binds Fc
Intended reaction
Binds antigen
polystyrene surface
Heterophilic antibody
Binds animal antibody in
a constant region
Human antimouse
antibody (HAMA)
Flow Cytometry
Cell suspension
Dichroic
mirrors
APC
or
Argon
laser
To Computer
PerCP
PE
FITC
sheath
fluid
Fluorochromes:
FITC: fluorescein isothiocyanate
PE: phycoerythrin
PerCP: peridinin chlorophyll
APC: allophycocyanin
charging collar
deflection plates
collection tubes
waste
Forward- and Side-Scatter Analysis
• Lymphocytes: smaller size,
no granules, spherical
nucleus. Low complexity.
• Monocytes: larger size, some
granules, indented nucleus.
Some complexity.
• Granulocytes: medium size,
many granules, segmented
nucleus. Most complexity.
• Data for individual
populations can be selected
and analyzed separately by
computer: “gating”
Single/Dual Parameter Plots
• Incubate cells with FITC-labeled anti-CD3
and PE-labeled anti-CD4 antibodies.
• Collect flow cytometry data.
• Gate on lymphocytes.
• Top figure: histogram for CD3 only differentiates between T cells and other
lymphocytes
• Bottom figure: dot plot for CD3 and CD4
- differentiates between helper T cells
and other T cells.
1% (0.3%)
52% (15.6%)
19% (5.7%)
28% (8.4%)
• % of lymphocyte gate
• (% of total leukocytes)
Pathologies caused
by antibodies
Waldenstrom’s Macroglobulinemia
• Proliferation of IgM-secreting cells.
• Tumors localize in lymphoid organs (enlarged lymph nodes and spleen)
• Increased blood viscosity impedes blood flow through vessels.
• The IgM paraproteins can behave as cryoglobulins that precipitate (or
agglutinate with RBC) and occlude small vessels in extremities during
cold weather (Raynoud phenomenon).
• Neuropathies can develop if
IgM attacks peripheral nerves.
• Vasculitis develops if IgM is
directed against IgG.
Impaired renal function
Systemic Lupus Erythematosis
Goodpasture’s
“lumpy-bumpy” immune
complexes trapped by
filtration
“linear” antibody deposition
on glomerular basement
membranes
Autoantibodies attack receptors
motor neuron
Hypothalmus
TRH
Pituitary
TSH
ACh
B
AChR
A
Graves’
disease
stimulatory
inhibitory
Na+
skeletal muscle fiber
Thyroid
Myasthenia gravis
Autoantibody detection
Fluorescence Antinuclear Antibody (ANA) Test
(for systemic autoimmune diseases)
Fix HEp-2 (human epithelial cell line) on microscope slide
Plasma membrane
Nuclear membrane
Permeabilize
Add patient serum
(with autoantibodies)
• >95% of SLE patients have
a positive ANA, but other
conditions (e.g., RA),
and even some healthy
individuals, can be positive
• Titers >80 are usually
reliable indicators of SLE
Wash, add fluorescent
anti-Ig, wash
ANA staining patterns
homogeneous
interphase
metaphase
rim
speckled
nucleolar
centromere
Homogeneous
Histone
Diagnosis
Coarse
Confirmatory
testing for specific
autoantibodies
ANA
Pattern
Diagnostic decisions using ANA
DIL
dsDNA*
PCNA
Speckled
Fine
Coarse
SSA/Ro
SSB/La
RNP
high
titer
SS
MCTD
Sm
DNP
Atypical
SCL-70
Nucleolar
Centromere
none
none
RNP
Systemic lupus
erythematosus
Scleroderma
*Most specific for SLE, can also cause a rim (peripheral) pattern
DIL: drug-induced lupus, SS: Sjogren’s syndrome, MCTD: Mixed connective tissue disease
DNP: deoxyribonucleoprotein, PCNA: proliferating cell nuclear antigen, RNP: ribonucleoprotein
Antibodies in viral diagnosis
Typical Time Course for Viral Infection
Incubation
days-weeks
Relative
magnitude
of response
Acute Infection
weeks-months
anti-viral
IgM
viral
antigen
Recovery
weeks-months-years
anti-viral IgG
symptoms
Time
• Earliest indicators: cultured virus or viral antigen
• IgM: only present during or soon after infection
• IgG: measure soon after onset of symptoms, then 10-30 days later 4x increase will indicate recent infection
• Newborns: Must measure IgM, IgG could be maternal
Hepatitis B time course (acute infection)
Incubation
8-13 weeks
Acute Infection
2 weeks-3 months
Early Recovery
3-6 months
Full Recovery
>6 mo-years
total anti-HBc
anti-HBs
HBsAg
Relative
magnitude
of response
anti-HBe
HBeAg
anti-HBc
IgM
symptoms
Time
• Anti-HBs is the only serological marker in a vaccinated individual.
• Patients can be treated by passive transfer of immune globulin.
Hepatitis B (chronic infection)
Incubation
8-13 weeks
Acute Infection
2 weeks-3 months
Symptoms subside
2-4 months
HBsAg
Relative
magnitude
of response
Chronic carrier
total anti-HBc
anti-HBe
HBeAg
anti-HBc
IgM
symptoms
Time
• HBsAg remains in serum
• Anti-HBs not produced in patients who become chronically infected.
• HBeAg may or may not be present, depending on stage of disease
(it indicates active viral replication).
Epstein-Barr virus diagnosis
• Heterophile antibodies are produced that react with horse, sheep and
cow (bovine) red blood cells.
◦ Monospot test: serum agglutination to horse RBC.
◦ Paul-Bunnell test: serum agglutination to sheep RBC.
◦ Replaced by latex agglutination or point-of-care
immunochromatographic tests using purified antigens.
• For symptomatic patients with negative heterophile antibody results
(10-15%), retest for anti-EBV antibodies:
◦ ELISA using several recombinant EBV antigen for capture (easier),
- or ◦ Indirect immunofluorescence assay using EBV-infected cells
(“gold standard”)
EBV Time Course
Antigen titer
Primary infection
Convalescence
Reactivation
Early Antigen
Viral Capsid Antigen
EBV Nuclear Antigens
Symptoms
Symptoms
VCA IgM
Antibody titer
VCA IgG
Heterophile
IgM
EBNA IgG
VCA IgM
EA IgG
~2 months
Time
EA IgG
months, years later
Laboratory Testing: Antibody to HIV
• Standard screening test: hybrid ELISA, solid phase coated with several
HIV-1 and HIV-2 proteins, and antibody against p24. Bound antibodies
detected with labeled HIV antigens, bound p24 detected with labeled
antibody.
◦ detects antibodies of several isotypes
◦ ability to detect p24 allows earlier diagnosis
• If positive → retest twice by same ELISA
• ELISA has low positive predictive value in low-risk populations (only
13% of positives are actually infected)
• If one/both of retests are positive → confirm, usually by western blot
Western blot for detecting antibody to HIV
• Western blot has several antigens applied to membrane
(separate tests for HIV-1 and HIV-2).
• Profile of antibodies indicates stage of infection:
◦ p24 and p55: appear early, then fade
◦ gp41, gp120, gp160: appear later, sustained
throughout disease
• Western considered positive if two of the following bands are present: p24, gp41 and gp120/160.
• Can be read visually or quantified with a densitometer.
• High positive predictive value > 99%.
Administering antibodies
to patients
Immunolocalization
• Intravenous injection of low-intensity
radiolabeled monoclonal antibody to
image metastasis.
Liver
• Example: Prostascint, which is an
antibody against prostate-specific
membrane antigen (PSMA) labeled
with 111indium.
• Prostate must be removed first.
• Arrows indicate tumor metastasis to
lymph nodes.
• Large mass is the liver, which is
clearing the compound.
Radioimmunotherapy
• Intravenous injection of high-intensity
radiolabeled monoclonal antibody to kill tumor.
B
CD20
• Example: Bexxar (antibody against CD20
conjugated with 131iodine) is a treatment for
B-cell non-Hodgkin’s lymphoma.
• Unlabeled antibody given first to reduce
binding to normal B cells in spleen.
• Bexxar administered, tumor cells destroyed,
but normal B cell count is reduced.
Immunotherapy
Passive immunotherapy
• Infusion of monoclonal antibody against tumor antigen.
• Example: Herceptin interferes with the human epidermal growth
factor receptor-2 (HER-2), inhibiting tumor growth.
Active immunotherapy
• Vaccine (Gardasil) produced from purified, inactive human
papillomavirus is injected i.m. → primary immune response →
generate memory cells to combat subsequent infection of HPV, which
can cause cervical and vaginal cancers
Reducing Immunogenicity of Therapeutic
Antibodies
mouse
chimeric
humanized
Murine variable
domains grafted
onto human
constant domains
Murine hypervariable regions
grafted into
human variable
domains
human