Chapter 10

Flow Cytometry Assessment of Lymphocyte Populations

Infiltrating Liver Tumors
Maria Pérez-Lanzón, Céleste Plantureux, Juliette Paillet, Jules Sotty,
Patrick Soussan, Guido Kroemer, Maria Chiara Maiuri, and Jonathan Pol

Abstract
Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immu-
nosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune
checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such
research progress relies on the in-depth characterization of the immune populations that are present in
pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is
high-dimensional cytometry.
In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte
populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required
for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of
surface and intracellular staining of more than a dozen markers of interest. This protocol does not require
prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure
includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell
surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we
provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic
subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-
staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.

Key words Tumor immune microenvironment, Liver immunity, Tumor-infiltrating lymphocytes,

Hepatic lymphocytes, Flow cytometry, Single-cell analysis

1 Introduction

Hepatocytes require the heterogeneous compartment of non-

parenchymal cells (NPCs) to support their physiological function.
Almost half of NPC cells are leukocytes, with lymphocytes and
myeloid Kupffer cells as the main immune populations in both
humans and mice [1]. This healthy hepatic immune environment

Guido Kroemer et al. (eds.), Liver Carcinogenesis: Methods and Protocols,

Methods in Molecular Biology, vol. 2769, https://doi.org/10.1007/978-1-0716-3694-7_10,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

129
130 Maria Pérez-Lanzón et al.

is considered to be tolerogenic to avoid chronic inflammation due

to continuous exposure to external agents [2].
However, alterations of the hepatic immune environment
appeared in multiple pathological conditions, such as liver autoim-
mune diseases and cancer. In autoimmune hepatitis and primary
biliary cholangitis, self-reactive B and T lymphocytes attack
non-transformed hepatocytes [3] and cholangiocytes [4], respec-
tively. Moreover, the liver is exposed to immunomodulators
derived from infectious sources or from chronic inflammatory con-
ditions that may contribute to neoplastic transformation of hepa-
tocytes in hepatocellular carcinoma (HCC) or cholangiocytes in
cholangiocarcinoma (CCA) [5, 6], favored by the failure of natural
antitumor immunosurveillance [7–9]. This hepatic immune envi-
ronment is however therapeutically targetable, as demonstrated by
the clinical implementation of immune checkpoint blockers (ICBs)
for the care of HCC [10] and, more recently, CCA [11]. Indeed,
ICBs targeting the PD-1/PD-L1 interaction increased the survival
of patients with HCC in combination with anti-VEGF, and CCA in
combination with standard gemcitabine and cisplatin [10, 11].
The identification of immunotherapy targets benefits from the
in-depth characterization of (human or murine) liver immune
populations achieved by multiparameter single-cell assays. Among
such assays, flow cytometry (FC) allows to simultaneously study the
cell subtype, differentiation state, and functional polarization of
individual immune cells. Conventional FC is generally more afford-
able and user-accessible than spectral FC or cytometry powered by
time-of-flight (CyTOF), even though less cellular markers can be
covered [12–14].
Here, we present a protocol for characterizing the lymphocytic
populations infiltrating the liver by conventional FC in mice. Up to
13 cell surface and intracellular markers reflecting different pheno-
types and states of lymphocyte maturation at a single-cell level can
be assessed with an input of 100 mg of fresh non-perfused liver.
We made use of the protocol of hepatic leukocyte isolation
reported by Lambertucci et al. in Chap. 1 to obtain a cell suspen-
sion enriched in immune populations. This cell suspension can be
immediately transferred for surface staining or used for cell culture,
including non-specific or antigen-specific lymphocyte stimulation
[15]. Once the individually separated cells are available, the cell
suspension can be directly transferred into a U-shaped tube or
96-well plate for FC staining. Removal of debris and dead cell by
gradient cell separation or specific enrichment kits is not required.
Indeed, debris and dead cells are gradually eliminated during the
tissue processing steps and will ultimately be discarded from analy-
sis due to the gating strategy applied for FC data analysis (Fig. 2).
Of note, it is recommended to work using 96-well plates to carry
 Flow Cytometry Assessment of Hepatic Lymphocytes 131

out parallel staining of multiple samples using time-effective multi-

channel pipets and minimal volumes to achieve a reduction of
reagents and costs.
In brief, the protocol includes the following sequence of
events: (i) staining of damaged cells, (ii) blocking of Fc receptors
(FcR) to prevent non-specific antibody binding, (iii) targeting sur-
face markers of interest with specific fluorescent antibodies, (iv) cell
fixation as end- or pause-point prior to (v) additional staining of
intracellular/nuclear (ICN) markers (Fig. 1). The 13-plex FC anti-
body panel illustrated in the present chapter targets both cell
surface and nuclear proteins and allows to discriminate the main
subsets of lymphocytes (i.e., conventional and regulatory CD4+ T
cells, CD8+ T cells, TCRγδ+ T cells, NK cells, or B cells) and their
differentiation states (naı̈ve, central memory [Tcm], effector mem-
ory [Tem], or effector [Teff] T cells) (Table 1, Fig. 2) [16–18].
Although this antibody panel does not include the assessment of
intracellular cytokine production [15], the protocol includes an
ICN staining in which supplementary antibodies specific to such
protein targets could be incorporated, particularly if the laboratory
is equipped with a spectral flow cytometer.

Liver single Antigen-specific (or not)

cell suspension ex vivo cell restimulation

Viability dye

FcR blocking

Cell surface (CS) staining

(antibody cocktail 1)

Wash

Cell fixation

Wash +
Cell permeabilization
STOP point CS
PAUSE point ICN
Intracellular / nuclear (ICN)
staining (antibody cocktail 2)

Wash
STOP point ICN

Acquisition on flow cytometer

Data analyses

Fig. 1 Overview of the cell surface and intracellular/nuclear staining procedure of

liver cell suspensions. Pause and stop points are depicted
132 Maria Pérez-Lanzón et al.

Table 1
Panel of dye and antibodies used for phenotyping hepatic lymphocyte subsets

Target Clone Dye/Fluorochrome Proposed Concentration (ng/μL)

Viability dye
Dead cells – Fixable yellow LIVE/DEAD (dilution at 1:500)
Fc receptor blocking
CD16/CD32 2.4G2 – 2.5
Surface staining
CD4 GK1.5 BUV496 0.25
CD45 30-F11 BUV661 1
CD3e 145-2C11 BV421 2
NK1.1 PK136 BV605 2
TCRgd GL3 BV711 2
CD25 PC61 BV786 2
CD19 1D3 PerCP-Cy5.5 0.5
CD8a 53–6.7 PE 0.5
CD27 LG.3A10 PE-Dazzle594 2
CD62L REA828 PE-Vio770 3
CD44 IM7 APC 2
B220 REA755 APC-Vio770 0.75
Intracellular/nuclear staining
FoxP3 FJK-16 s FITC 10

The use of a fixative-resistant cell viability dye, which allows cell

fixation after surface marker staining, facilitates the introduction of
an end- or pause-point in which ICN staining can be postponed up
to the next day. If the latter is not required, samples can be directly
stored after fixation for up to one month at 4 °C until data acquisi-
tion on a flow cytometer. Alternatively, cell fixation can be skipped
if the samples are acquired immediately post-staining without
compromising cell viability. Finally, we provide an example of a
gating strategy for the retrieval of qualitative and quantitative
information about the lymphocyte populations detectable in liver
cell suspensions stained with the abovementioned panel (Fig. 2)
[16–18].
We successfully applied this protocol to orthotopic HCC [19]
(Fig. 2) and CCA [16] (data not shown), induced by systemic
injections of diethylnitrosamine or oncogene-encoding plasmids,
as reported by Sotty et al. in Chap. 2, and Plantureux et al. in
Chap. 8 of this book, respectively.
 Flow Cytometry Assessment of Hepatic Lymphocytes 133

A Cells Single cells CD45+

Viability dye
FSC-H
SSC-A

CD45
FSC-A FSC-A FSC-A FSC-A

Living CD45+ CD3+ NK1.1- CD3+ CD4+ CD8- CD3+ CD4+ CD8- CD44+ CD62L-

CD62L
FoxP3

CD27
CD3

CD4

NK1.1 CD8 CD25 CD44 FSC-A

CD3+ CD4- CD8- CD3+ CD4- CD8+ CD44+ CD62L-

CD27
CD62L
TCRgd

FSC-A CD44 FSC-A

CD3- NK1.1-
CD19

B220

B Markers for gating strategy ( living cells) Cell type

CD45+ Leukocytes
CD45+ CD3- NK1.1+ NK cells
CD45+ CD3+ NK1.1- T cells
CD45+ CD3+ NK1.1- CD8+ CD4- CD8+ T cells
CD45+ CD3+ NK1.1- CD8- CD4+ CD4+ T cells
CD45+ CD3+ NK1.1- CD8- CD4+ FoxP3+ CD4+ regulatory T cells
CD45+ CD3+ NK1.1- CD8- CD4- TCRgd + TCRgd + T cells
CD45+ CD3- NK1.1- CD19+ B220+ B cells
CD45+ CD3- NK1.1- CD19- Myeloid cells
For CD4+ and CD8+ T cells
CD44- CD62L+ Naïve T cells (Tn)
CD44+ CD62L+ Central memory T cells (Tcm)
CD44+ CD62L- CD27low Effector memory T cells (Tem)
CD44+ CD62L- CD27- Effector T cells (Teff)

Fig. 2 (a) Example of a gating strategy used to identify the indicated lymphocyte populations in a freshly
dissociated bulk liver sample of DEN-CCl4-induced orthotopic HCC using the antibody cocktails indicated in
Table 1. (b) Phenotype of the main lymphocytic cell subsets
134 Maria Pérez-Lanzón et al.

2 Materials

2.1 Materials and 1. Aluminum foil.

Equipment 2. Filter 0.22 μm.
3. Adapted syringe (optional, for filter 0.22 μm).
4. Tubes for reagent preparation (50 mL or 1.5 mL).
5. U-shaped 96-well plate or 5 mL tubes for FC staining and
running.
6. Adhesive foil.
7. Multichannel pipets (optional).
8. Reservoirs (optional).
9. Fridge.
10. Rocket platform (optional).
11. Vortex (optional).
12. Centrifuge.
13. Chemical hood.
14. Flow cytometer.
15. FC analysis software.

2.2 Reagents 1. Bovine Serum Albumin (BSA).

2. Sterile phosphate buffer saline (PBS) 1X (without calcium and
magnesium, pH = 7.4).
3. Sterile deionized distilled water (ddH2O).
4. Fixative-resistant cell viability dye.
5. FcR blocking antibodies α-CD16/α-CD32.
6. Antibody cocktail solution for the desired panel.
7. Antibody buffer (if required for the antibody cocktail) (i.e., BD
Horizon™ Brilliant Stain Buffer, BD Biosciences, Ref:
563794).
8. Fixative solution (optional) (i.e., BD Cytofix/Cytoperm™,
BD Biosciences, Ref: 554714) for staining of surface antigens
or eBioscience™ Foxp3/Transcription Factor Staining Buffer
Set (Thermofisher, Ref: 00–5523-00) for staining intra-cellu-
lar/nuclear antigens.
9. Permeabilization/Washing (PW) solution (optional) (i.e., BD
Perm/Wash™ Buffer, BD Biosciences, Ref: 554723).
10. FC compensation beads (optional).
 Flow Cytometry Assessment of Hepatic Lymphocytes 135

3 Methods

3.1 Preparation of The following buffer can be prepared the day before the experiment
Buffers_Time: 10 min to speed up the staining procedure, and thus preserve cell viability.
1. Prepare FC buffer by dissolving BSA at 0.5% (w/v) in PBS
under sterile conditions. To accelerate BSA dissolution, you
can use a vortex or leave the solution on a rocker platform.
Then, filter the FC buffer through a 0.22 μm filter (coupled to
an appropriate syringe, if necessary) and store at 4 °C until use.
Example: For 50 mL of FC buffer, add 50 mL of PBS to 250 mg of
BSA.

3.2 Preparation of 1. Resuspend each cell pellet of dissociated liver in a desired

the Single Cell volume of cold PBS according to the initial weight of tissue.
Suspension for Immu- We recommend a concentration of 500 mg liver per mL. Pipet
nostaining_Time: up and down until sample homogeneity is reached.
1 min per Sample (See 2. For each FC staining panel, transfer the desired volume of liver
Notes 1–4) cell suspension into a 96-well round bottom plate (hereafter
referred to as FC staining plates) (see Notes 5 and 6). If
necessary, complete with PBS to achieve a final volume of
200 μL of liver cell suspension per well. We recommend stain-
ing 100 mg of liver per FC panel, that is, 200 μL of the cell
suspension at 500 mg/mL (see Note 7).

3.3 Cell Surface 1. Centrifuge the plate for 5 min at 400 g at 4 °C.
Immunostaining_ 2. In the meantime, prepare fixative-resistant cell viability dye
Time: 150 min per solution (viability solution) for step 4 as follows. Dilute the
Sample (but viability dye in the solvent recommended by the manufacturer.
Simultaneous Sample The total volume must be sufficient to add 100 μL per well at
Staining Is Possible) the concentration established from your titration results
(See Notes 8–11) (Table 1). Store at 4 °C until use protected from light in
aluminum foil.
3. Discard the supernatant by plate reversion. In more detail,
reverse the plate quickly and firmly on top of a liquid waste
container then, while keeping the plate upside down, slightly
press it against a paper tissue to remove liquid excess before
placing it back on the bench.
4. Add 100 μL of viability solution to each cell pellet and resus-
pend by pipetting gently. Incubate for 15 min at 4 °C protected
from light in aluminum foil.
5. In the meantime:
(a) Prepare the FcR blocking solution for step 9. To do so,
dilute α-CD16/α-CD32 antibody in FC buffer in order
to have 100 μL of FcR blocking solution per well
(Table 1).
136 Maria Pérez-Lanzón et al.

(b) Prepare the antibody cocktail solution for surface staining

occurring in step 13. To do so, dilute your surface
marker-specific antibodies according to the titration
results in FC buffer (see Note 12) in order to have
100 μL of antibody cocktail solution per well (Table 1).
6. After incubation, add 100 μL of PBS to all wells to achieve a
final volume of 200 μL/well.
7. Centrifuge the plate for 5 min at 400 g at 4 °C.
8. Discard the supernatant by plate reversion.
9. Resuspend each cell pellet in 100 μL of FcR blocking solution
by pipetting gently. Incubate for 10 min at 4 °C protected from
light in aluminum foil.
10. After incubation, add 100 μL of PBS to all wells to reach a final
volume of 200 μL/well.
11. Centrifuge the plate for 5 min at 400 g at 4 °C.
12. Discard the supernatant by plate reversion.
13. Resuspend each cell pellet in 100 μL of surface marker-specific
antibody cocktail solution by pipetting gently. Incubate for
25 min at 4 °C protected from light in aluminum foil (Table 1).
14. After incubation, add 100 μL of FC buffer in each well to reach
a final volume of 200 μL/well.
15. Centrifuge the plate for 5 min at 400 g at 4 °C.
16. In the meantime, prepare (if needed) a fixative solution under a
chemical hood following the manufacturer’s recommendations
(see Note 13).
17. Discard the supernatant by plate reversion.
18. Wash each cell pellet by resuspending them in 200 μL of FC
buffer.
19. Centrifuge the plate for 5 min at 400 g at 4 °C.
20. Discard the supernatant by plate reversion.
21. Under a chemical hood, resuspend each cell pellet in 100 μL of
the fixative solution. Incubate for 15 min at 4 °C protected
from light in aluminum foil (see Note 14).
22. In the meantime, prepare 1X Perm/Wash (PW) washing solu-
tion in ddH2O or according to the manufacturer’s instructions
(see Note 15).
23. After incubation, add 100 μL of PW solution to all wells to
reach a final volume of 200 μL/well.
24. Centrifuge the plate for 5 min at 400 g at 4 °C.
25. Discard the supernatant by plate reversion.
 Flow Cytometry Assessment of Hepatic Lymphocytes 137

26. Wash all cell pellets by resuspending them in 200 μL of PW

solution.
27. Centrifuge the plate for 5 min at 400 g at 4 °C.
28. Discard the supernatant by plate reversion.
29. Repeat the previous three steps (steps 26–28). If intracellular
staining is not required, proceed to step 8 of Subheading 3.4.
If required, prepare in the meantime the antibody cocktail
solution for ICN staining (Table 1) and proceed to
Subheading 3.4.

3.4 Intracellular/ 1. Resuspend each cell pellet in 100 μL of ICN staining antibody
nuclear Flow cocktail solution per well. Incubate for 25 min at 4 °C pro-
Cytometry tected from light in aluminum foil.
Staining_Time: 60 min 2. After incubation, add 100 μL of PW solution to all wells to
per Sample (but reach a final volume of 200 μL/well.
Simultaneous Sample 3. Centrifuge the plate for 5 min at 400 g at 4 °C.
Staining Is Possible)
4. Wash each cell pellet by resuspension in 200 μL of PW solution.
5. Centrifuge the plate for 5 min at 400 g at 4 °C.
6. Discard the supernatant by plate reversion.
7. Repeat the three previous steps (steps 4–6).
8. Resuspend all cell pellets in 200 μL of FC buffer per well by
pipetting gently. Seal the plate with an adhesive foil and store it
at 4 °C, protected from light, until acquisition on a flow
cytometer.

3.5 Quick Overview Acquire samples in a flow cytometer with lasers and filters adapted
of Sample Acquisition to your selected fluorochromes. For the lymphocyte antibody panel
Through a Flow that we propose here, the calculation of a compensation matrix for
Cytometer, Analysis, fluorochromes with an overlapping fluorescence spectrum is
and Cell Count required. To calculate compensations, you will need
Normalization (i) monostained samples (i.e., a set of individual samples each
stained with one single antibody or dye of the panel), (ii) a fully
3.5.1 Sample Acquisition stained sample (i.e., which allows detection of all the markers
Through Flow targeted by the panel), and (iii) an unstained sample. The latter
Cytometer_Time: 120 min samples can be cells (of the tissue of interest or of a positive control
for Compensations and tissue) or commercially available compensation beads. For samples
5 min per Sample Acquired monostained with the viability dye, a fraction of both living and
(See Note 16) dead cells is required. Refer to your flow cytometer software
instructions to adapt laser voltages to your fluorochromes and
calculate the corresponding compensation matrix. This matrix can
be modified during post-acquisition analyses for all samples
recorded.
138 Maria Pérez-Lanzón et al.

3.5.2 Analyses and Cell Generated files (commonly of the .fcs type) can be analyzed using
Count Normalization_Time: open-access resources (i.e., FlowSOM for R studio) or commercial
120 min per Sample (but softwares (e.g., BD FACS DIVA™, FlowJo™, and Omiq). Most
Simultaneous Sample analyses rely on initial phenotyping of cells by lineage markers
Analysis Is Possible) (See followed by the characterization of specific markers associated
Note 17) with cell activation, exhaustion, or differentiation, among others
(Fig. 2). These analyses can be user-supervised (Fig. 2) or semi-
supervised depending on the analytical tool use. The most common
result outputs include total counts of cells, percentages of cells
according to a parent population, or measures of the median/
mean fluorescent intensity for each specific marker.
To convert relative (raw) cell counts given by the flow cyt-
ometer to absolute counts, we recommend a normalization step
taking into account the initial liver weight and measurements of the
volume of cell suspension prior to and after acquisition on the
cytometer. To do so, apply the following formula:
Absolute cell count normalized per mg of liver = Raw cell count
x Factor 1 x Factor 2.
In which:
Factor 1 = total weight of initial liver/actual weight of liver
transferred for FC staining. For this point, consider the weight of
liver transferred for FC staining in Subheading 3.2.
Factor 2 = (volume before cytometer – volume after cyt-
ometer)/volume before cytometer.

4 Notes

1. It is important to measure the liver weight before tissue pro-

cessing and dissociation. Our protocol is optimized for staining
100 mg of liver with each antibody cocktail.
2. We follow the protocol presented by Lambertucci et al. in
Chap. 1 to obtain a single-cell suspension from bulk
non-perfused livers. As previously mentioned, this protocol is
optimized for the recovery of leukocytes, especially for flow
cytometry purposes. Be aware that the viability of other cell
populations (e.g., hepatocytes) may be compromised.
3. We recommend including a positive control tissue according to
your antibody panel (i.e., spleen for lymphocyte populations)
to verify the quality of the staining and help to guide post-
acquisition analyses.
4. All along the protocol, products were used at room tempera-
ture unless otherwise indicated.
5. If the number of samples to analyze is limited, U-shaped 5 mL
tubes for FC can be used as an alternative to plates.
 Flow Cytometry Assessment of Hepatic Lymphocytes 139

6. Save an unstained aliquot of the homogenized tissue and, if

enough material is available, another aliquot to perform single
staining with the fixative-resistant cell viability dye. These sam-
ples will be useful for setting up compensations on the flow
cytometer and guiding post-acquisition analyses.
7. The fraction of bulk cell suspension chosen for staining will be
considered for data normalization in Subheading 3.5.2.
8. The flow cytometry staining procedure described here is opti-
mized for the use of plates. If tubes are preferred, indicated
volumes of cell resuspension may need to be adapted.
9. We take advantage of the periods of incubation to prepare the
solutions needed for the subsequent step. Alternatively, these
solutions can be prepared in advance prior to achieving Sub-
heading 3.3. In our hands, the preparation of solutions 3 hours
before use did not have any experimental impact.
10. All fluorescent antibodies or dyes should be titrated according
to the tissue analyzed and your experimental purpose. Some
suggested concentrations are indicated in Table 1.
11. Conjugated antibodies coupled to fixative-sensitive fluoro-
chrome tandems such as APC-Cy7 or PE-Cy7 are not recom-
mended for this protocol since they can be cleaved during the
fixation step, thus causing detectable non-specific signal in the
APC and PE channels, respectively.
12. Some of the fluorochromes used in our antibody panel require
the addition of BD Horizon™ Brilliant Stain Buffer (50% v/v)
(BD Biosciences, Ref: 563794) in addition and prior to the
incorporation of the mix of antibodies and FC buffer.
13. We recommend adapting the fixative solution to the purpose of
the staining performed. Staining of intracellular or nuclear
(ICN) antigens requires previous cell permeabilization,
whereas this step is not necessary for the staining of cell surface
antigens.
14. Short incubation time in the fixative solution helps to prevent
cleavage of fluorochrome tandems.
15. PW solution may contain saponin to wash the cells while
allowing permeabilization for ICN staining if needed.
16. The time indicated is estimated for the acquisition of the
lymphocyte antibody panel presented here. It can vary accord-
ing to the size of the panel of antibodies and the cell subsets
analyzed.
17. The time indicated is estimated for the analyses of the lympho-
cyte antibody panel presented here using supervised analysis by
a software-familiarized user following the gating strategy of
Fig. 2.
140 Maria Pérez-Lanzón et al.

Acknowledgments

J.G.P. is supported by the SIRIC Cancer Research and Personalized

Medicine (CARPEM); Multi-Organism Institute (ITMO) Aviesan
Cancer (National Alliance for Life Sciences and Health), Institut
National du Cancer (INCa), and Fondation pour la Recherche
Médicale (FRM). M.C.M. is supported by the SIRIC Cancer
Research and Personalized Medicine (CARPEM). G.K. is sup-
ported by the Ligue contre le Cancer (équipe labellisée); Agence
National de la Recherche (ANR) – Projets blancs; AMMICa
US23/CNRS UMS3655; Association pour la recherche sur le
cancer (ARC); Cancéropôle Ile-de-France; European Research
Council Advanced Investigator Grant “ICD-Cancer,” FRM; a
donation by Elior; Equipex Onco-Pheno-Screen; European Joint
Programme on Rare Diseases (EJPRD); European Research Coun-
cil (ICD-Cancer), European Union Horizon 2020 Projects Onco-
biome and Crimson; Fondation Carrefour; Institut National du
Cancer (INCa); Institut Universitaire de France; LabEx Immuno-
Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE
Award from the Mark Foundation; the RHU Immunolife; Seerave
Foundation; SIRIC Stratified Oncology Cell DNA Repair and
Tumor Immune Elimination (SOCRATE); and SIRIC Cancer
Research and Personalized Medicine (CARPEM).

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