on the agenda is a brief overview of the advanced high throughput flow cytometer
the IQ platform which was used to create the data scene in this webinar I will then give an
insight into how T-cell exhaustion occurs and how this progresses specifically with
regard to an oncology setting and how the tumor micro environment plays a role here
finally I will cover two case studies the first looking at byte antibodies and their effect on
T Cell exhaustion compared to a more rudimentary activation method and the second will cover how
the T-cell exhaustion kit provided by Sartorius can allow us to identify car T Cell Activation
the iq3 is an advanced flow cytometer capable of reading from either 96 or 384 well plates at high
speed the iq3 also allows a vast quantity of information to be collected from up to
15 detection channels the optimized assays and reagents provided by Sartorius require a small
volume of cells and supinating and therefore minimize the amount of pressure sample required
the iq3 system comes with built-in foresight software allowing for
fast time to results as grass Auto populate as cells are being sampled
as I've mentioned the iq3 has a suite of compatible reagents that are applicable to a range
of different applications namely within the immuno oncology antibody Discovery and foundational areas
the iq3 allows a user to simultaneously test T Cell phenotype alongside secreted proteins in
a 10 microliter sample from a 96 or 384 well plate this provides insight into the status
of multiple cell types within a complex cell environment such as the quantification of cv3
positive lymphocyte populations live and dead cell readouts and also cell marker expression
Q beads included in the wells allow the capture and quantification of multiple specific protein
or cytokine readouts such as tnf Alpha and granzyme B I will now begin to discuss how
T-cell exhaustion comes about within an oncology setting T cells within the tumor microenvironment
interact with antigen presenting cells or apcs which present the cells with a foreign antigen
this stimulates the T cells to activate via the T Cell receptor pathway and or CD3
activation of T cells induces low-level upregulation of the pd1 and lag-3 markers to both
control the magnitude of the initial activation and also to act as a negative feedback loop
upon activation the T cells produce a number of cytokine and chemokines to stimulate other
immune cell types to respond the activated T cells also act by killing the cancerous cells
over time antigen presenting cells begin to produce inhibitory signals
tumor cells also release inhibitory factors into the tumor microenvironments such as tryptophan
other cells in the tumor microenvironments such as tumor-associated fibroblasts inhibit
T cell function and activation via faceligand accumulating inhibitory signals and long-term
exposure to the same antigen cause increasing inhibitory receptors to
be present on the T Cell these include pd1 lig3 ctla4 fast Tim 3 and many more
as the tumor begins to escape the immune system and proliferate mounting inhibitory signals are
produced increased complexity forms within the tumor microenvironment is increasing inhibitory
cells types infiltrate such as tumor-associated macrophages these further Aid the tumor in its
growth this blocks appropriate T Cell response such as cytotoxicity proliferation and cytokine
production this loss of effector function is otherwise known as T Cell exhaustion
Sartorius have reduced the T-cell exhaustion cell and cytokine profiling kit which is available for
violet blue red laser IQ and iq3 systems this reagent kit allows for the quantification of
viability proliferation T Cell subtype CD3 CD4 and cd8 T Cell status markers pd1 lag 3 and Tim 3 and
also cytokine secretion of interferon gamma and tumor necrosis Factor Alpha all from a single well
cytokine standards are provided to allow quantification of protein production and
pre-defined compensation settings allowed the user to quickly ascertain their results
also provided with the T-cell exhaustion kit is the predefined gating strategy
this speeds up the time to results as these gates are auto-populated whilst the data is acquired
foresight also allows the fine-tuning of these Gates by using positive and negative populations
initially Q beads and cells are clearly separated using the forward scatter and side scatter graph
singlet beads and cells are then selected for and Q beads are then split into tnf Alpha and
interferon gamma based on the beads internal fluorescence the single cells can then be split
into effector and B Green encoded target cells if target cells are not used then the effector
cell may be encoded with the B Green encoder die provided with the kit viability of effector
cells can then be deduced and live effectors can be taken forward into the phenotype Gates
CD3 cells are first identified and these are further phenotyped
into CD4 Helper and cd8 cytotoxic populations
finally pd1 lag 3 and team 3 positive cells can be identified in each of these groups
to First evaluate T Cell exhaustion CD3 positive T cells were treated in one of the following
three ways non-stimulated shown by the blue histogram Peak stimulated once with dynabead CD3
cd28 represented in light gray or stimulated with dynabeads repeatedly every two to three days for a
period of seven days shown in dark gray dynabeads activate T cells via the CD3 T Cell receptor
pathway and also using a co-stimulatory signal via anti-cd28 mimicking the interaction in Vivo with
antigen presenting cells here we show a series of histograms of pd1 expression on CD3 positive cells
Gates were set based on the day Zero populations as you can see after two days both of the
stimulated populations increase in pd1 expression indicated with a right shift in the histogram at
day five we see the single stimulated population decrease I.E shift to the left while the repeat
stimulation maintains its pd1 expression finally a day 7 the single stimulation returns
back to non-stimulated levels while the repeat stimulation sees sustained expression of pd1
the same repeat stimulation was used to pre-exhaust CD3 positive cells in this assay
non-exhausted cells from the same donor we use as a comparison here the cells were plated in well
and treated once on Day Zero with a dose response of dynabeads CD3 to cd28 a 10 microliter sample
was taken on three sequential days from the same plate and analyzed for expression of exhaustion
markers and cytokines on day one sixty percent of exhorted cells Express pd1 irrespective of the
concentration of dynabeads used to re-stimulate them lag 3 positive expression is also very high
in exhausted cells ranging from between 80 to 100 percent the non-exhausted cells exhibit low levels
of both pd1 and lag-3 on day one dependent on the density of Dino beads they were stimulated with
day two shows a similar pattern with increasing levels of exhaustion markers on non-exhausted
cells while those that were pre-exhausted maintain high and consistent pd1 and lag-3 expression
by day three non-exhausted cells match or exceed the exhausted
population when looking at both pd1 and lag 3 expression at high densities of dynabeads
when looking at the full-time course it is clear that pd1 expression decreases slightly in
pre-exorted cells re-stimulated with low densities of dynabeads however this is not the case with
lag 3 where expression is sustained at all stimulation concentrations in pre-exhausted cells
cytokines from the same assay plate were also assessed non-exhaustive cells show concentration
dependent increase in the production of tnf Alpha from day one in contrast exhausted
T cells are produced significantly reduced concentrations of tnf alpha over the time course
the highest bead stimulation using 200 000 dynabeads per well cause non-exhausted
cells to produce 12-fold more tnf Alpha than pre-exhausted cells with the same stimulation
tnf Alpha production Peaks at 0.7 nanograms per ml when exhausted cells are treated with 12 500
dynabeats per well reduce tnf Alpha production is a Hallmark of T-cell exhaustion and this
reduction can easily be detected and Quantified alongside marker expression using this kit
I will now move on to how bi-specific T-cell engage antibodies function
and cover some data as to how this affects the exhaustion phenotype scene
bite antibodies are made up of fragments from two antibodies attached by a hinge
that function by binding to both the T cell and the target cell simultaneously
this brings the activated T Cell into direct contact with a specific Target cell of Interest
in this case study we've used an anti-cd3 cross cd19 bite antibody this exclusively binds to cd19
positive target cells such as those in B cell lymphomas allowing specific Target cell killing
once the antibody has bound to both Target and the T Cell the T Cell undergoes clonal
expansion and proliferation while the target cell is attacked and eventually will undergo apoptosis
this anti-cd3 cross cd19 antibody was used in an immune cell killing assay set up in
comparison with a more crude method of T Cell Activation namely dynabead CD3 cd28
Nook like green labeled Ramos target cells we use as our cd19 positive
Target cell line and pbmc is isolated from Whole blood we use as our effector cells
after 60 hours in plate a 10 microliter sample was taken and analyzed with the T-cell exhaustion kit
as you can see pd1 expression is concentration dependent with both activator types but is
expressed at a higher level in dynabeads compared to the CD3 cd19 bite this can also
be reviewed directly from foresight software in the form of a heat map shown on the right
lag 3 also follows this trend with higher expression seen and pbmcs activated with
dynabeads compared to bite at higher speed densities we see 25 of CD3 cells expressing
lag-3 compared to just 12 when treated with the highest concentration of bite 10 nanograms per ml
team 3 expression is relatively low across all treatment types and highlights the temporal
nature of these exhaustion markers whereby pd1 is expressed shortly after activation followed
by lag 3 and finally Tim 3 after high levels of stimulation to the same antigen
we're looking overall at the levels of exhaustion markers one might assume that because the
exhaustion levels are higher in the dynabead wells that more killing will have occurred here
however the count of live target cells was also collected from the T Cell exhaustion cell and
cytokine kit by using the nut like green labeled Ramos cells these can easily be separated from
the effector cells using the bl1 versus side scatter height Dot Plot as shown on the right
as you can see at highest concentrations of byte we see a 31 decrease in the number
of target cells compared to the highest dynabeat density
the Insight achieved from multiple aspects of the kit allows for the identification of
more specific activators that may enhance our ability to tackle specific genotypes in Vivo
this case study highlights how the bite antibody induces lower pd1 and lag free expression after 60
hours in co-culture showing the more specific and gentle activation approach causes lower
levels of exhaustion despite enhanced killing of specific target cells when compared to dynabeads
the final case study utilizes anti-cd19 chimeric antigen receptor T cells or car
T cells to Target a cd19 positive b-cell lymphoma
these car T cells have been engineered to specifically identify cd19 on a Target cell
when this occurs the T Cell will activate and undergo rapid clonal expansion and attack the cd19
positive cell causing tumor cell death however upon interaction with a cd-19 negative Cell No
Such proliferation nor attack should occur this makes these cells highly specific and useful in
a clinical setting to Target cancerous cells while leaving antigen negative healthy cells untouched
we compared car T cells to non-activated and dynabeat activated T cells obtained from the
same donor over the course of three days the car T cells used are 54 antigen positive meaning
46 of cells within the car T worlds are normal non-transfected T cells effector cells were plated
in the presence of cd19 positive Ramos nut green cells or cd19 negative yurkatina green cells 10
microliter samples were taken on three sequential days and analyzed using the T Cell exhaustion kit
when looking at tnf Alpha and interferon gamma production it is clear that across
the three days there was little response when non-activated T cells were present
T cells with activation however so increased cytokines over the three days interestingly
this was higher in the presence of Ramos cells than your cat cells in both cytokine readouts
when looking at the car T Cell response we see high levels of cytokine production but
only in the presence of the cd19 positive Target cell line showing
that the car T cells are responding and activating in response to these cells
furthermore we utilize the multiple metrics obtained by the multiplex IQ kit to assess
effector cell proliferation and Target cell count non-activated affected do not exhibit
much proliferation over the three days and the same is true for the activated T cells
Target cell proliferation is also apparent in Worlds with non-activated cells however when
activated T cells are included we see enhanced killing of your cat cells as this is preliminary
data we are still looking to complete further studies to elucidate why this is occurring
when looking at the car T data we see significant car T proliferation in the presence of the Ramos
cell line again suggesting the car T cells are activating in response to the cd19 positive
stimuli corresponding decline in Ramos cells compared to the non-actuated control is also seen
these results further identify the enhanced activation that car
T cells are undergoing in the presence of their target cell
the expression of pd1 lag3 and tim3 was also collected however due to the early time points
of this data set we are showing pd1 data as an early marker of activation in non-activated T
cells we again see little to no pd1 expression across the three-day time period while activated
T cells show maintained pd1 expression around 10 percent in the presence of both target cells
again we see a difference when car T cell effectors are present with an increased pd1
expression in the presence of Ramos cells when compared to low non-activated levels of pd1 in
the presence of cd19 negative year cats in summary tnf Alpha and interferon gamma cytokine release
shows the car T cells are activating in response to the cd19 antigen positive cell line car T Cell
proliferation is higher in Wells with Ramos cells compared to cd19 earcat cells a decrease in total
viable cd19 positive target cells can be seen highlighting the specific killing of the Ramos
cells early exhaustion marker pd1 is also more highly expressed when grammar cells are present
this case study helps to identify an alternative use of the T-cell exhaustion kit highlighting that
it can be used to First identify activation of effector cells before later time points identify
the exhaustion of cells by decreased cytokines and increase late exhaustion marker Tim III
to summarize the IQ Advanced flow cytometry platform and its compatible reagents allows the
simultaneous detection of cells and cytokines from a single 10 microliter sample per well
utilizing the human T Cell exhaustion cell and cytokine profiling kit
allows successful identification of exhausted self-enotypes which can be
tracked over a period of days to allow a timeline of events to be elucidated
Therapeutics or Gene edited cells of interest can be used in the presence of a Target cell type
this allows greater insight into the specificity and function of these potential treatment types