Professional Documents
Culture Documents
Flow Cytometry Assessment of Lymphocyte Populations Infiltrating Liver Tumors
Flow Cytometry Assessment of Lymphocyte Populations Infiltrating Liver Tumors
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.
1 Introduction
129
130 Maria Pérez-Lanzón et al.
Viability dye
FcR blocking
Wash
Cell fixation
Wash +
Cell permeabilization
STOP point CS
PAUSE point ICN
Intracellular / nuclear (ICN)
staining (antibody cocktail 2)
Wash
STOP point ICN
Data analyses
Table 1
Panel of dye and antibodies used for phenotyping hepatic lymphocyte subsets
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
CD27
CD62L
TCRgd
B220
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
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.
3.3 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.
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
Acknowledgments
References
1. Racanelli V, Rehermann B (2006) The liver as hepatocellular carcinoma. Nat Rev Gastroen-
an immunological organ. Hepatology 43:S54 terol Hepatol 188(18):525–543
2. Thomson AW, Knolle PA (2010) Antigen- 9. Loeuillard E, Conboy CB, Gores GJ et al
presenting cell function in the tolerogenic (2019) Immunobiology of cholangiocarci-
liver environment. Nat Rev Immunol 10:753– noma. JHEP Rep 1:297–311
766 10. Llovet JM, Castet F, Heikenwalder M et al
3. Mieli-Vergani G, Vergani D, Czaja AJ et al (2021) Immunotherapies for hepatocellular
(2018) Autoimmune hepatitis. Nat Rev Dis carcinoma. Nat Rev Clin Oncol 193(19):
Prim 4 151–172
4. Ma WT, Chen DK (2019) Immunological 11. Greten TF, Schwabe R, Bardeesy N et al
abnormalities in patients with primary biliary (2023) Immunology and immunotherapy of
cholangitis. Clin Sci (Lond) 133:741–760 cholangiocarcinoma. Nat Rev Gastroenterol
5. Yang JD, Roberts LR (2010) Hepatocellular Hepatol 2023:1–17
carcinoma: a global view. Nat Rev Gastroen- 12. Nolan JP, Condello D (2013) Spectral flow
terol Hepatol 7:448–458 cytometry. Curr Protoc Cytom. Chapter 1
6. Karlsen TH, Folseraas T, Thorburn D et al 13. Bendall SC, Nolan GP, Roederer M et al
(2017) Primary sclerosing cholangitis – a com- (2012) A deep Profiler’s guide to cytometry.
prehensive review. J Hepatol 67:1298–1323 Trends Immunol 33:323
7. Schreiber RD, Old LJ, Smyth MJ (2011) Can- 14. Gadalla R, Noamani B, MacLeod BL et al
cer immunoediting: Integrating immunity’s (2019) Validation of CyTOF against flow cyto-
roles in cancer suppression and promotion. metry for immunological studies and monitor-
Science 331:1565 ing of human cancer clinical trials. Front Oncol
8. Sangro B, Sarobe P, Hervás-Stubbs S et al 9:415
(2021) Advances in immunotherapy for 15. Pol JG, Bridle BW, Lichty BD (2020) Detec-
tion of tumor antigen-specific T-cell responses
Flow Cytometry Assessment of Hepatic Lymphocytes 141
after oncolytic vaccination. Methods Mol Biol during Leishmania major infection requires
2058:191–211 IL-12 to produce IFN-γ. J Immunol 180:
16. Paillet J, Plantureux C, Lévesque S et al (2021) 8299–8305
Autoimmunity affecting the biliary tract fuels 19. Uehara T, Pogribny IP, Rusyn I (2014) The
the immunosurveillance of cholangiocarci- DEN and CCl4 -induced mouse model of
noma. J Exp Med 218 fibrosis and inflammation-associated hepato-
17. Martin MD, Badovinac VP (2018) Defining cellular carcinoma. Curr Protoc Pharmacol
memory CD8 T cell. Front Immunol 9:2692 66:14.30.1–14.30.10
18. Pakpour N, Zaph C, Scott P (2008) The cen-
tral memory CD4+ T cell population generated