Rationale
The broad goal of our research is the generation of organs-on-chip
models of human lymphoid tissue to study human immunology and
immunotherapy. In this proposal, we use design principles from the human
lymph node and the clinically targeted receptor CTLA-4 as a case study
to identify the impact of providing relevant 3D organization, matrix and
cell types on human immunology R&D. The lymphoid tissue module will be
invaluable in
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Mimicking polarization of the T and B cell receptor machinery and the
presence of Fc-receptor expressing cells to analyze the true efficacy
and cytokine release induced on cross-linking of therapeutic
antibodies. This includes immediate antibody mediated cytokine release
syndrome (e.g. rituximab) and delayed immune related adverse events
seen with checkpoint blockade (4).
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Recapitulating 3D interactions between human immune cells, cancer
cells and human endothelial cells-important for tumor induced
immunosuppression, trafficking of anti-tumor lymphocytes (including
CAR T cells) and antibodies (e.g rituximab efficacy on malignant B
cells resident in the LN) and their function.
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Using the prototype human lymphoid tissue on chip (hLToC) to design
in vitro trials for some of the staggering number of
combinatorial immunotherapies being considered for cancer treatment
and identification of the best combinations for clinical trial.
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Using the hLToC to uncover personalized tumor-immune biology and test
the impact of genetic diversity on anti-tumor immunity.
There are two published 3D models of human lymphoid tissue, both
trying to recapitulate the human lymph node: VaxDesign’s MIMIC system
and Probiogen’s HuALN (8). In the
HuALN, the T and B cells are disorganized. The HuALN shows long-term
survival and generation of IgM in response to antigen but no IgG,
suggesting that the immune response does not mature. In the MIMIC
system, T and B cells cultured on separate microcarriers are mixed to
simulate T cell dependent antibody responses. There is no reticular
network and immune response is generated by immigrant dendritic cells.
The model doesn’t recapture LN anatomy and has not been tested for
immunooncology.
The design principles we have followed derive from a
Preliminary data:
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Use of matrix and cell populations based on published data:
The extracellular matrix (ECM) composition of human LNs is unknown but
there are preliminary studies on primates. We compared existing
literature about the primate LN ECM
(9) and Matrigel
(10). Matrigel is a commercially
available basement membrane-like gel that contains a fibrillar network
of collagen IV, as well as laminin and heparin sulfate proteoglycan,
which is reminiscent of the primate LN ECM. To improve longevity of
the gel, we added 1.5mg/ml bovine collagen I. PBMC preparations have
40-50% T cells and only 3-15% B cells. Whole lymph nodes, however,
contain ~50-60% T cells and ~40-50%
B cells. The previously published MIMIC system has shown that cultures
of T and B cells in a LN-like ratio produce higher titers of Ig
(8). Thus in preliminary studies
we cultured an approx. 1:1 mix of T and B cells in 60%Matrigel and
1.5mg/ml collagen I.
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Polarization of CD3 and CTLA4 upon 3D culture: We found that
4-7 days of culture, a significant fraction of T cells were polarized
with CD3 and CTLA-4 accumulating in cap like structures.
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Lymph-tissue-on-chip
design: We engineer microfluidic devices of different geometries with
polydimethylsiloxane (PDMS), an optically clear, biocompatible and
stable polymer. These devices can be perfused for weeks via engineered
vessels lined with human vascular or lymphatic endothelium and
visualized in-situ by microscopy; it is also possible to perfuse these
devices with whole human blood. We can engineer ring-like (Fig 2A) or
parallel channels (Fig 2B) to mimic separated T and B zones as seen in
the LN and perfuse them via lymphatic and blood vessels (ideal for
studying trafficking into the LN). Yet, in many tertiary lymphoid
structures, this segregation is not as evident and we started our
experiments with a thin mixed lymphocyte gel (T+ B, 200uM thickness)
separated from a perfusion channel by a porous membrane (Fig. 2C).
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Controlling
for donor and experimental variability: Tertiary lymphoid structures
can be quite variable. Thus for establishing donor and experimental
variability, we relied on design principles from the LN and basic
lymphocyte activation assays. A LN-like T:B ratio of 60:40 in standard
2D cultures was activated with heat-killed formalin fixed Staph.
aureus Cowan I (SAC) (Fig. 3). SAC is commonly used to model
T-cell dependent polyclonal immunoglobulin (Ig) production, and it is
typically performed using purified B cells provided with T cell
produced cytokine or using whole PBMC preparations that have 40-50% T
cells and only 3-15% B cells. Whole lymph nodes, however, contain
~50-60% T cells and ~40-50% B cells.
The previously published MIMIC system has shown that cultures of T and
B cells in a LN-like ratio produce higher titers of Ig
(8).
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Use of Matrigel based on published data: The extracellular
matrix (ECM) composition of human LNs is unknown but there are
preliminary studies on primates. We compared existing literature about
the primate LN ECM (9) and
Matrigel (10). Matrigel is a
commercially available basement membrane-like gel that contains a
fibrillar network of collagen IV, as well as laminin and heparin
sulfate proteoglycan, which is reminiscent of the primate LN ECM.
Thus, in preliminary studies, we cultured lymphocytes in Matrigel. In
future studies several biological and synthetic polymers will be
tested.
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Organoid
formation in Matrigel: The T and B cells isolated from human PBMC
were labeled with two different colors of CellTrace or CellTracker
dyes to enable discrimination. Over multiple donors, we defined
labeling conditions and the right consistency of Matrigel. We expected
that uniform distribution of lymphocytes would be maintained in the
absence of organizing cues from stromal cells. Serendipitously we
found that in 2-4 days, the cells organize into clusters, in
particular the B cells form clusters in a bed of T cells (Fig. 4A and
B), reminiscent of B cell follicles in the LN. Cluster formation was
density dependent. 1-2 million cells in 10ul formed clusters, but 0.5
million cells did not (Fig. 4C), suggesting close range or
contact-dependent interactions. Clustering occurred in experiments
performed both on chip and in Transwell membrane cultures. Our data
suggest that T and B cells can drive their own 3D organization. This
phenomenon is especially relevant to the formation of tertiary
lymphoid organs which develop without the presence of lymphoid tissue
inducer (LTi) cells known to important in embryonic and neonatal
lymphoid organization. Previous data indeed suggest that B cells can
perform LTi like functions in tertiary lymphoid tissue
(11).
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IL-2
production by hLN-on-chip: To simulate antigen arrival at the
organized structures, we will fabricate microfluidic chips containing
channels lined by human lymphatic endothelial cells. However, we first
confirmed that antigen can be delivered under flow to the chip. We
perfused 1:14,000 SAC in the perfusion channel and were able to
stimulate IL-2 production (Fig. 5).
These data demonstrate that an organ-on-chip strategy is feasible to
create an in vitro models of human lymph tissues. Further we were
able to establish the survival and function of key lymphocyte
populations. We have also optimized labeling of the immune cell
recovered from PBMC such that they can be visualized for 2 weeks by
imaging.
C. Specific aims: Cancer immunotherapy can be best designed by
using human tissue provided with the right biomechanical cues. Yet,
peripheral blood mononuclear cells (PBMC) are the most commonly
available resource. Our preliminary data provides evidence that
development of a prototype human lymphoid module using engineered
polydimethylsiloxane microfluidic devices and human peripheral blood
mononuclear cells is feasible. The human lymphoid tissue on chip (hLToC)
will enable the study cancer immunotherapy in an entirely human, patient
specific paradigm. To focus our studies on a discrete lymph node
function and benchmark the impact of increasing matrix and cellular
complexity on immunotherapy design, we chose to study the expression
pattern of CTLA-4, a clinically relevant target and response to CTLA-4
blockade therapy. The matrix composition that we have defined in
preliminary experiments induces CD3 polarization as seen in the human
lymph node where T cells are primed for response. As predicted, we find
that CTLA-4 is also polarized. In doing so, our preliminary data
identified an intriguing phenomenon: the self-organization of
lymphocytes into CTLA-4+ clusters. Thus our specific aims are:
Normal donors:
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Test standard immune responses in in a 3D microfluidic model of human
lymphoid tissue (hLT-on-chip) and describe the efficacy and toxicity
of CTLA-4 blockade
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Perfusion of the hLT-on-chip with lymphatic and blood endothelial
cells via microengineered vessels and impact on function and response
to CTLA-4 blockade
Cancer patients:
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Modeling the impact of cancer on human lymphoid tissue, specifically
lung cancer, lymphoma and chronic lymphocytic leukemia
D. Research Design and Methods
Aim 1: Test standard immune responses in in a 3D microfluidic
model of human lymphoid tissue (hLT-on-chip) and describe the safety
and efficacy of CTLA-4 blockade
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T and B cell stimulation will be studied using
anti-CD3 antibody OKT3, which is known to stimulate T cells in PBMC
cultures and with heat-killed formalin fixed Staph. aureus
Cowan I (SAC) which induces polyclonal Ig production to test if the 3D
organization improves reactivity to immunostimulation. OKT3 is
particularly relevant as it is an immunomodulator used to prevent
graft rejection and induces cytokine release as seen with anti-cancer
antibodies. In addition to providing the ability to activate B cells,
SAC serves as positive control due to the abundance of pathogen
associated molecular patterns (PAMPs, eg: LPS). The parameters tested
will be IL-2 and IgM production by ELISA, dilution of the
fluorophore as cells divide and lastly multiplex cytokine assays to
perform unbiased assessment of functional states via differences in
cytokine profile.
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Are the CTLA4+ clusters germinal centers-AID staining
and IgG production: The clusters that we observe in response to SAC
stimulation have densely packed B cells along with CTLA4+ T cells.
This is reminiscent of germinal center staining in human lymph nodes.
This presents the intriguing possibility that we have modeled the
germinal center in vitro. The key distinguishing factor between
2D stimulation of peripheral B cells vs. in-vivo responses is the
appearance of germinal centers is enhanced class switching and somatic
hypermutation. We will test these by checking the expression of AID
(expressed during class switching and somatic hypermutation),
….(expressed only during somatic hypermutation) and checking
the level of IgG production. If we can detect…, we might
further perform immunosequencing i.e. testing the sequences of the
antibodies where divergence of genomically encoded sequences can be
detected. In case we do NOT observe somatic hypermutation, our model
is still valuable to assess the impact of T cell polarization, matrix
and tissue like density on immunotherapeutic responses.
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Effect of anti-CTLA-4 therapy on T and B cell
responses: hLN-on-chip prototype will be activated with OKT3 and SAC
with or without monoclonal antibodies blocking CTLA-4 and these data
will be compared to T cell activation in 2D culture with bead bound
antibodies. Readouts defined in (b) will be tested. To our knowledge,
these data would be the first entirely human, in vitro model of
CTLA-4 blockade in previously untreated tissue resident T-cells.
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Providing a reticular network of antigen capturing
cells: The lymph node stroma is composed of several types of immune
and stromal cells that form a reticular network continuously scanned
by lymphocytes for antigen and costimulatory signals
(12). Further subcapsular sinus
macrophages provide a layer of retained antigen for lymphocytes to
access. To model retained antigen and costimulatory signals provided
by non-lymphocytes within organized tissue, we have 3 accessible cell
types in monocytes (CD14+ magnetic bead selection before isolation of
lymphocytes) and monocyte derived macrophages and dendritic cells
(moDC).
In preliminary experiments we will first use monocytes and monocyte
derived dendritic cells (GM-CSF/IL-4 cultured monocytes) to establish
an antigen presenting stroma for lymphocyte scanning. Depending on the
success of these experiments and if time permits, the other cellular
sources we can explore are monocyte derived macrophages, macrophages,
dendritic and stromal cells from the human lymph node. We have already
tested that a 5% monocyte frequency can be successfully introduced in
the gel. We expect to test a range within 5-20% and ask if
self-clustering, polarization and response to stimulation and
checkpoint blockade changes in single cell suspension recovered from
the hLN-on chip. Using the assays described above, we will test the
impact of Mo/Mo-DC inclusion on the response to SAC, OKT3 and
anti-CTLA-4, alone or in combination.
Caveats: If organization of PBMC into lymphoid tissue
like structures needs to be optimized, we can use bioengineering
approaches to create cytokine microdomains or use lentiviral
overexpression of cytokines. It is possible that the clusters we
observe do not show AID positivity and somatic hypermutation-we will
again learn from in-vivo design and ask what other cell types,
secreted signals and matrix components can be provided to induce
somatic hypermutation in response to activation. Further, not all
tertiary lymphoid structures show germinal center activity. Thus our
model could still be used to test the impact of immunotherapy of
activation of lymphocytes within tumor associated tertiary lymphoid
structures.