Nature,
Год журнала:
2021,
Номер
596(7870), С. 126 - 132
Опубликована: Июль 21, 2021
Abstract
PD-1
blockade
unleashes
CD8
T
cells
1
,
including
those
specific
for
mutation-associated
neoantigens
(MANA),
but
factors
in
the
tumour
microenvironment
can
inhibit
these
cell
responses.
Single-cell
transcriptomics
have
revealed
global
dysfunction
programs
tumour-infiltrating
lymphocytes
(TIL).
However,
majority
of
TIL
do
not
recognize
antigens
2
and
little
is
known
about
transcriptional
MANA-specific
TIL.
Here,
we
identify
clones
using
MANA
functional
expansion
assay
3
neoadjuvant
anti-PD-1-treated
non-small
lung
cancers
(NSCLC).
We
use
their
receptors
as
a
‘barcode’
to
track
analyse
coupled
single-cell
RNA
sequencing
receptor
sequencing.
find
both
MANA-
virus-specific
TIL,
regardless
response,
MANA-,
influenza-
Epstein–Barr
each
unique
programs.
Despite
exposure
cognate
antigen,
express
an
incompletely
activated
cytolytic
program.
hallmark
tissue-resident
memory
(TRM)
cells,
low
levels
interleukin-7
(IL-7R)
are
functionally
less
responsive
(IL-7)
compared
with
influenza-specific
TRM
cells.
Compared
from
responding
tumours,
non-responding
tumours
markedly
lower
ligand-dependent
signalling,
largely
confined
HOBIT
high
subsets,
coordinately
upregulate
checkpoints,
killer
inhibitory
inhibitors
activation.
These
findings
provide
important
insights
overcoming
resistance
blockade.
Nature Communications,
Год журнала:
2019,
Номер
10(1)
Опубликована: Окт. 17, 2019
Human
T
cells
coordinate
adaptive
immunity
in
diverse
anatomic
compartments
through
production
of
cytokines
and
effector
molecules,
but
it
is
unclear
how
tissue
site
influences
cell
persistence
function.
Here,
we
use
single
RNA-sequencing
(scRNA-seq)
to
define
the
heterogeneity
human
isolated
from
lungs,
lymph
nodes,
bone
marrow
blood,
their
functional
responses
following
stimulation.
Through
analysis
>50,000
resting
activated
cells,
reveal
signatures
mucosal
lymphoid
sites,
lineage-specific
activation
states
across
all
sites
including
distinct
for
CD8+
an
interferon-response
state
CD4+
cells.
Comparing
scRNA-seq
profiles
tumor-associated
our
dataset
reveals
predominant
compared
within
multiple
tumor
types.
Our
results
therefore
establish
a
high
dimensional
reference
map
health
analyzing
disease.
Science Immunology,
Год журнала:
2019,
Номер
4(34)
Опубликована: Апрель 5, 2019
The
discovery
of
T
cells
resident
in
diverse
tissues
has
altered
our
understanding
adaptive
immunity
to
encompass
site-specific
responses
mediated
by
tissue-adapted
memory
throughout
the
body.
Here,
we
discuss
key
phenotypic,
transcriptional,
and
functional
features
these
tissue-resident
(TRM)
as
established
mouse
models
infection
translated
humans
novel
tissue
sampling
approaches.
Integration
findings
from
human
studies
may
hold
unlocking
potential
TRM
for
promoting
preventing
infection.
Annual Review of Immunology,
Год журнала:
2019,
Номер
37(1), С. 521 - 546
Опубликована: Фев. 6, 2019
Resident
memory
T
(Trm)
cells
stably
occupy
tissues
and
cannot
be
sampled
in
superficial
venous
blood.
Trm
are
heterogeneous
but
collectively
constitute
the
most
abundant
cell
subset.
form
an
integral
part
of
immune
sensing
network,
monitor
for
local
perturbations
homeostasis
throughout
body,
participate
protection
from
infection
cancer,
likely
promote
autoimmunity,
allergy,
inflammatory
diseases
impede
successful
transplantation.
Thus
major
candidates
therapeutic
manipulation.
Here
we
review
CD8
European Journal of Immunology,
Год журнала:
2017,
Номер
47(10), С. 1584 - 1797
Опубликована: Окт. 1, 2017
The
marriage
between
immunology
and
cytometry
is
one
of
the
most
stable
productive
in
recent
history
science.
A
rapid
search
PubMed
shows
that,
as
July
2017,
using
"flow
immunology"
a
term
yields
more
than
68
000
articles,
first
which,
interestingly,
not
about
lymphocytes.
It
might
be
stated
after
short
engagement,
exchange
wedding
rings
officially
occurred
when
idea
to
link
fluorochromes
monoclonal
antibodies
came
about.
After
this,
recognizing
different
types
cells
became
relatively
easy
feasible
only
by
simple
fluorescence
microscope,
but
also
complex
sometimes
esoteric
instrument,
flow
cytometer
that
able
count
hundreds
single
second,
can
provide
repetitive
results
tireless
manner.
Given
possibility
analyse
immune
phenotypes
variety
clinical
conditions
has
changed
use
cytometer,
which
was
incidentally
invented
late
1960s
measure
cellular
DNA
intercalating
dyes,
such
ethidium
bromide.
epidemics
HIV/AIDS
1980s
then
gave
dramatic
impulse
technology
counting
specific
cells,
since
it
clear
quantification
number
peripheral
blood
CD4+
T
crucial
follow
course
infection,
eventually
for
monitoring
therapy.
As
consequence,
development
cytometers
had
easy-to-use
all
laboratories
helped
widely
disseminate
this
technology.
Nowadays,
rare
find
an
immunological
paper
or
read
conference
abstract
authors
did
main
tool
dissect
system
identify
its
fine
functions.
Of
note,
developments
have
created
sophisticated
mass
cytometry,
simultaneously
dozens
molecules
at
cell
level
allows
us
better
understand
complexity
beauty
system.
However,
moon
dark
side.
strengths
technology,
i.e.
fact
often
brief
training
sufficient
start
producing
data,
weakness.
Indeed,
several
(too
many)
papers,
eye
well-trained
cytometrist
aspects
would
need,
polite,
"little"
improvement.
Not
mention
cases
technical
mistakes
are
performed,
involving,
among
others,
(in)adequate
controls,
(lack
appropriate)
compensation,
sorting
strategies,
even
description
methods
used.
For
reason,
editorial
team
European
Journal
Immunology
feels
worthwhile
offer
our
community
guidelines
correct
cytometric
techniques
field
immunology.
Thus,
starting
Congress
(ECI
2015)
Vienna
(Austria)
under
guidance
Professor
Andreas
Radbruch,
we
asked
colleagues
friends,
renowned
field,
contribute
sharing
their
knowledge
particular
areas
expertise,
order
present
collection
protocols
great
interest.
Such
information
includes,
suggestions
tricks
regarding
how
study
phenotypes,
type
amount
produced
secreted
stimulation
population
interest,
signalling
processes,
differentiation,
proliferation
death,
cytotoxic
activities,
cell-cell
interactions,
activity
intracellular
organelles
mitochondria,
response
induced
against
tumours
anticancer
immunosuppressive
drugs,
transcription
factor
activity,
soluble
molecules,
drug
uptake,
events.
Today's
challenges
involve
choice
reagents,
preparation
eventual
storage
analysis,
overall
experimental
plan
and,
last
least,
data
analyses.
We
no
longer
limited
instrumentation,
creativity
ask
critical
questions.
These
"Guidelines
studies"
thus
represent
effort
collect
currently
accepted
best
variation
major
players
(along
with
functionality)
include
standards
interpretation,
well
cautions
issues.
One
aspect
concerns
reproducibility,
topic
recently
attracted
considerable
attention.
Therefore,
meant
help
researchers
avoid
potential
pitfalls
could
drastically
alter
interpretation
data.
While
preparing
guidelines,
feedback
received
feel
should
highlighted
Introduction.
Firstly,
"FACS"
(fluorescence
activated
sorting)
used
Becton
Dickinson
(BD)
technologies
BD
trademark
(FACSTM);
general
sorting"
company
agnostic.
Secondly,
CD
mAbs
anti-CD
(in
other
words
CD1
mAb
anti-CD1
mAb,
example)
This
because
nomenclature
primarily
cluster/characterize
later
describe
respective
molecules.
Thirdly,
although
comprehensive
possible,
there
naturally
limitations
e.g.
subset
antigens
shown
times,
certain
reagents/companies
examples.
opinion
efforts
must
improved—this
science
works!
glad
receive
from
readers
comments,
new
ideas,
articles
possible
future
updates
Guidelines.
Before
closing,
like
thank
four
people
who
played
role
ensuring
Radbruch's
Andrea
Cossarizza's
vision
reality.
Hyun-Dong
Chang
Ute
Hoffman,
both
DRFZ,
Karen
Chu,
former
Associate
Editor,
Cate
Livingstone,
Managing
Editor
Immunology.
Together
core
coordinated
author
invitations,
submission,
peer
review
revision
sections
proofs,
sought
incorporated.
full
invaluable
work
on
project.
To
accurately
optical
properties
pass
through
uniformly
bright
center
focused
laser
beams.
Light
optics
intersection
point
beams
pick
up
scattered
light
cells.
sensing
zone
here
measurements
parameters
taken.
In
stream-in-air
sorters,
located
around
0.3
mm
nozzle
tip,
inside
cuvette.
purpose
fluidic
move
precisely
region
liquid
stream
way
each
illuminated
same
lasers.
sort
capabilities
establish
break
off
small
uniform
droplets.
Droplets
containing
interest
charged
deflected
electric
sorting.
kind
technique
Mack
J.
Fulwyler
1965
Los
Alamos
National
Laboratory
1.
needed
machine
testing
performance
Coulter
counters,
so
particle
separator
particles
volumes.
Len
Herzenberg
interested
living
basis
fluorescence,
he
got
design
plans
found
little
group
Stanford
University
build
FACS
(see
video
Inventing
Cell
Sorter,
Lab,
https://www.youtube.com/watch?v=Ro8P3w9BPhg).
precise
positioning
jet
hydrodynamic
focusing
counters
2.
suspension
injected
thin
tubing
laminar
sheath
fluid
enters
wide
into
narrow
orifice.
speeds
diameter
sample
(sample
core)
decreased
(Fig.
1).
Crosland-Taylor
described
Nature
1953
3
device
suspended
fluid.
Some
years
before
1947,
F.T.
Gucker
similar
detecting
bacteria
air
4.
takes
place
so-called
chamber
cytometer.
detailed
optimized
sorter
patent
applications
Gerrit
van
den
Engh
5,
6
cuvette
another
application
7.
addition
chambers
based
cytometers,
arc
lamp
source
were
developed.
early
standard
microscope
epi-fluorescence
setup.
Here
lens
bring
excitation
take
emission
Excitation
separated
dichroic
mirror
special
filters.
With
immersion
high
numerical
aperture,
stabilized
staining
protocol,
histograms
coefficient
variations
(CVs)
lower
1%
(0.50–0.7%)
achieved
8,
9.
technique,
aligned
precision
micrometer.
rates
increased,
however,
out
focus
laser.
get
illumination.
means
accuracy
lost.
loss
measurement
increases
maintain
intensity,
elliptical
spots.
Typical
sizes
spot
60–150
micrometers
horizontally
5–20
vertically.
Recently,
beam
shaping
flat
top
introduced
manufacturer.
intensity
profile
gaussian
60
100,
150
micrometer
diameters
Fig.
approximation
calculation
ten
times
concentration
three
bigger
necessary
keep
rate.
fluid,
PBS
(phosphate
buffered
saline)
filtered
0.22
0.1
filter
compatible
species
sorted.
An
acoustic
developed
Gregory
Kaduchak
co-workers
2001
11,
12.
implemented
support
focusing.
helps
increase
if
cores
According
manufacturer,
acoustic-assisted
run
samples
low
concentrations
10
faster
compared
without
still
measurements.
fundamentals
given
13.
Based
invention
Richard
Sweet
14,
droplet
formation
vibrations
ultrasonic
transducer.
Little
disturbances
surface
exit
orifice
generated
grow
exponentially
lead
droplets
2,
10.
sorted
measured
moves
down
breakoff
point.
During
separation
jet,
voltage
pulse
jet.
So
electrons
caught
cannot
go
back
shut
off.
static
two
deflection
plates
3).
important
process
right
charge
droplet.
delay
charging
determined
operator
electronics.
done
beads
plates.
illuminates
streams
un-deflected
direction,
camera,
observed
monitor.
observation
spots
drop
brightness
maximized
minimized.
distance
controlled
held
constant.
setting
fixed
during
kept
constant
operator.
If
velocity
works
fine,
practice
always
case.
Small
changes
pressure
example
due
partial
clogging
Timothy
Petersen
examined
problem
showed
disturb
handle
16.
Toralf
Kaiser
temperature
alters
gives
solution
stabilizing
17.
schematic
typical
From
view
detection
capable
photons
wavelengths
over
dynamic
range.
achieve
range,
optics,
signal
detection,
processing
units
carefully
designed.
lenses
emitted
spatial
resolution
they
Furthermore,
make
collected
parallel
direct
bench
detectors.
employs
collimation
lenses.
Collection
(convex
lenses)
interrogation
either
end
fiber
directly
(e.g.
aspheric
condenser
lenses).
instruments
fibers
route
detected
detectors
installed
octagon.
case
ensure
routed
Inside
octagon
placed
front
detector
onto
photocathode.
photocathode
lens.
photodetectors
spectrally
broadband
therefore
unable
generate
exclusively
markers.
add
specificity,
filters
mirrors
defined
manner
Optical
designed
band
(BP),
long
(LP),
(SP)
mostly
common
property
transmit
within
spectral
BP
transmits
example,
named
660/20,
650
670
nm
will
photomultiplier
tube
reflected
next
set
specified
configuration.
SP
block
ones
whereas
LP
do
opposite
meaning
below
above
(LP)
wavelength.
660
nm.
Due
aging,
quality
coating,
contamination,
actual
parameter
differ
description.
recommended
check
transmission
spectra
provided
manufacturer
dust
free.
Sometimes
(usually
silver
mirrors)
deflect
geometrical
constructive
reasons.
>99%,
reflective
range
wavelengths.
contrast,
deflects
wavelength
while
rest
pass-through.
effect
dependent
operating
angle.
some
instruments,
dichroics
employed
working
angle
45°
others
12.5°.
commercial
become
available
spatially
dispersing
elements
instead
combination
array.
rationale
behind
entire
Section
I.3:
Flow
including
sorting).
element
dispersive
prism
grating.
Prisms
higher
efficiency
gratings
sensitive
polarized
light.
maybe
reason
why
Sony.
Lasers
mainly
solid-state,
continuous
wave
lasers
footprint
output
power
20
100
mW.
coherent
sources
allow
photon
density
illumination
point,
efficient
energy
transfer
fluorochrome.
Modern
equipped
seven
line
ranging
355
flexibility
choosing
fluorophores.
measures
biological
cell)
via
photons,
needs
converted
processed
amplifier,
filter,
analog
digital
converter
(ADC),
baseline
restorer
visualize
store
particles.
section,
components
electronics
briefly
described.
view,
related
difficult
(i)
level,
(ii)
analysis
rate,
(iii)
level.
Photomultiplier
tubes
(PMTs)
meet
these
requirements
almost
cytometers.
PMTs
vacuum
photocathode,
electron
electrodes,
series
dynodes
multiplication.
converts
photoelectrons
multiplied
driven
5).
Photocathodes
made
bialkali
material
determines
quantum
η
PMT,
ratio
incident
photons.
0<
<1
function
(h*f).
PMT
(R9220,
Hamamatsu)
=
0.2
500
0.09
700
reduction
sensitivity
7
dB.
PE-Cy7
less
FITC,
example.
many
applications,
increasingly
being
replaced,
avalanche
photodiodes
efficiency.
instrument
(CytoFlex,
Beckman
Coulter)
APDs
improve
>700
18.
All
amplifiers
analogue
hardware
devices
very
optimal
noise
ratios
(SNRs).
SNR
>86
Once
signals
pre-amplifiers,
amplifier
suitable
ADC
modern
conversion
discrete
values
ADCs
sampling
frequency
resolution.
required
(DNR)
stained
unstained
brighter
DNR
4
log
80
dB
(DNR[dB]=20log(104)).
ideal
by:
6.02*N
+
1.76
19.
theory
N
14
bit
86.04
practice,
effective
bits
is,
distortion
circuit,
decibels
theoretical
value
AD9240AS
Diva
electronic
78.5
20).
limits
decades
importantly,
shrinks
dim
signals.
MHz
30
per
speed
(pulse
length
μs).
peak
error
1–2%
21.
16
250
>4
<0.1%.
domain
filters,
restorer,
height,
width
algorithms,
trigger
Filtering
smoothen
raw
SNR.
resulting
consists
unwanted
DC
part
scatter
(among
others)
AC
part.
Hence,
subtracted
restorers
attempts
zero.
practise
restoring
perfect
negative
histogram
axis
introduce
slight
increased
CV
restoring,
(height,
width,
area)
extracted
*.fcs
file.
Taken
together,
algorithms
need
adjusted
maximize
DNR.
Since
prototype
Fluorescence
Activated
Sorter
1968
University,
powerful
analyze
individual
functional
status.
Moreover,
provides
robust
statistic
thousands
detect
events
10–4
uptake
multi-well
acquisition
rate
second.
decreases
increasing
cell.
Alternatively,
AttuneNXT
(ThermoFisher)
uses
keeping
tight
accurate
much
throughput.
serial
multiple
automated
high-throughput
platforms
(HyperCyt®).
Today,
27
bio-markers
Typically
markers
fluorescently
tagged
antibodies,
molecular
sensors,
genetically
encoded
reporters.
instance,
FACSymphony™
(Becton
Dickinson)
technically
50
achievable
moment
appropriate
fluorescent
dyes
limited.
Technical
maximum
detectable
overlap
tags,
channel
correlated
marker.
overcome
tags
Currently,
325
tags.
tunable
life
time
(FLIMs).
photomultipliers
diodes
convert
amplified
electrical
pulses
extract
area,
length,
time.
consist
basically
preamp
restoration
(ADC).
post-processing
(i.e.
integration,
log-transformation)
computer
software.
together
SNR)
(Q)
background
(B)
detection.
Avalanche
PMTs.
open
possibilities
long-wave
spectra.
CytoFLEX
(Beckman
Within
divided
division
multiplexer
(WDM)
integrated
array
enables
PE-Cy7.
itself
unsuitable
hence
mirrors.
chosen
multiparameter
experiment,
experiment
(markers)
analyzed,
requires
fluorophores
simultaneously;
consequence
spillover
III.1:
Compensation).
Conventional
circumvent
compensation
Compensation)
correlate
physical
Following
analyzed
multivariate
fashion
hierarchical
gating
strategy
VI.1:
Data
analysis—An
overview,
VI.2:
analysis—Automated
analysis:
Automated
identification
visualization).
essential
adapt
optical,
laser,
setup
minimize
spillover,
Q,
B
reagent
IV.2:
Titration—Determining
concentration),
fluorochrome
contributes
ideally
nothing
instrument.
(the
means)
blank
Q
B.
characterization
Mostly
polystyrene
(beads)
software
MACSQuant,
Fortessa,
Yeti,
Cytoflex
name
just
few.
Beads
say
"cell
dummies"
optimization,
setup,
counting,
scale
calibration
on.
Scale
especially
useful
approach
absolute
binding
photoelectrons)
relative
mean
intensities
(MFIs)
leads
quantitative
VII:
Cytometric
parameters).
Beside
beads,
LED
pulses.
quantiFlash™
(APE)
ultra
tool,
labs
values.
Up
analytical
been
can,
addition,
marker
expression
downstream
(molecular
biology,
sequ