Longitudinal tracking of T cell lymphomas in mice using flow cytometry

Summary

T cell hematological cancer has a complex interplay with host immune cells, but the ability to experimentally discriminate transferred cancer cells from host cells by flow cytometry is technically challenging. Here, we present a flow cytometry protocol to evaluate cancer cell and host immune phenotypes following transplant of a T cell lymphoma bearing a congenic marker (CD45.2) into a syngeneic host (CD45.1). We describe steps for isolation of primary immune cells from mice, staining preparation with flow cytometry antibody cocktails, and analysis by flow cytometry.

For complete details on the use and execution of this protocol, please refer to Kuczynski et al.¹

Graphical abstract

Highlights

• •Isolation of immune cells from murine spleens harboring transferred T cell lymphomas
• •Preparation of antibody stains to enable discrimination of host and cancer cells
• •Extracellular and intracellular antibody staining of immune cells
• •Flow cytometry acquisition and analysis using a BD LSRFortessa cytometer

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

T cell hematological cancer has a complex interplay with host immune cells, but the ability to experimentally discriminate transferred cancer cells from host cells by flow cytometry is technically challenging. Here, we present a flow cytometry protocol to evaluate cancer cell and host immune phenotypes following transplant of a T cell lymphoma bearing a congenic marker (CD45.2) into a syngeneic host (CD45.1). We describe steps for isolation of primary immune cells from mice, staining preparation with flow cytometry antibody cocktails, and analysis by flow cytometry.

Before you begin

The protocol below describes specific steps for evaluating mouse T-cell cancer cells and host immune populations by flow cytometry following adoptive transfer of a T-cell lymphoma bearing a monoclonal TCRvβ8 and expressing the congenic marker CD45.2 into a syngeneic host mouse bearing an alternative congenic marker CD45.1.¹ Cells are evaluated for a number of extracellular and intracellular markers through prosecution of two flow cytometry staining panels (staining panel A, and staining panel B). Elements of this protocols can be adapted in order to evaluate any transferred immune cell population (e.g., in the study of immune cell homeostasis, CAR T-cell biology, or immune cell fate tracking).^2,3 Moreover, these protocols can be applied for ex vivo evaluation of T-cell leukemia/lymphomagenesis models where cancer arises or is induced in situ.

In advance of the experiment, preparation of all buffers, pre-labeling of tubes and aliquoting reagents the day before beginning experimental work can greatly streamline the workflows, and minimize any user error.

Institutional permissions

In vivo studies were performed in the United Kingdom and Home Office approved them in accordance with the Animal Scientific Procedures Act 1986 (ASPA), IACUC guidelines, and AstraZeneca Global Bioethics policy. Studies were conducted on project licenses PCE886633 and P0EC1FFDF and were approved by the local AWERB committee. Animals were housed in compliance with Home Office guidelines.

Regulations in performance of animal procedure and in-life studies in mouse models varies between country and region. Care should be ensured that experiments are pre-approved and comply with national, regional and/or institutional guidelines. Studies should be performed with the 3 R’s principal in consideration⁴

Preparation of buffers

Timing: 1–2 h

• 1.
  Prepare required buffers.

  • a.
    Dissection medium: RPMI-1640 medium with 2% Fetal Calf Serum (FCS) and 0.02 M HEPES.

    • i.
      Add 10 mL FCS and 10 mL HEPES 1 M solution to 500 mL of RPMI-1640 medium.
  • b.
    Flow cytometry buffer: PBS, 0.1% Bovine serum albumin (BSA).

    • i.
      Add 0.5 g BSA to 500 mL of PBS. Stir, shake or rest until BSA has completely dissolved.
    • ii.
      Flow cytometry buffer can be stored at 4°C for 1–2 weeks. 0.09% Sodium Azide can be added optionally as a preservative.
  • c.
    Fixation and permeabilization reagents: Reconstitute components from the Invitrogen™ eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set according to manufacturer’s instructions (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2F00-5523.pdf).

    • i.
      Reconstitute Fix/perm buffer by diluting 1 part Fixation/Permeabilization Concentrate to 3 parts eBioscience Fixation/Perm Diluent. Make a final volume of 75 μL per sample to be analyzed + 10% overage.
    • ii.
      Dilute Permeabilization buffer 10× 1:10 in distilled water. Make a final volume of volume of 275 μL per sample to be analyzed + 10% overage.

Optional: Dialyzed lyophilized Collagenase type IV should be prepared freshly, by adding PBS to a final concentration of 5–10 mg/mL in PBS.

Note: Prepare buffers in advance and store at 4°C.

Note: If cells are being prepared from non-lymphoid tissue, or tissues require collagenase or enzymatic treatment to liberate cells, the addition of 2 mM Ethylenediaminetetraacetic acid (EDTA) to Flow Cytometry buffer can prevent clumping of dead cells during flow cytometry staining preparation. A limitation of this approach is that EDTA may influence some cellular proteins/epitopes by scavenging cationic co-factors which can change protein conformation. Care should therefore be taken to validate antibody staining fidelity in the presence of EDTA.

Note: Enzymatic preparations from alternative manufacturers can be considered to liberate immune cells from tissues. Cell numbers and population frequency may be impacted by the tissue preparation methods, and care should be taken to ensure consistency across experiments and groups.

Prepare working antibody cocktails

Timing: 30–60 min

• 2.
  Prepare working antibody staining cocktails/dilutions & label tubes appropriately.

  • a.
    Live/dead blue stain.

    • i.
      Reconstitute LIVE/DEAD Fixable Blue Dead Cell Stain Kit in Dimethyl sulfoxide (DMSO) according to manufacturer’s instructions (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Flive_dead_fixable_dead_cell_stains_man.pdf). Dilute a working concentration 1/1000 in PBS. Stain volume should be 100 μL/ experimental sample +10% overage.
  • b.
    Staining panel A (extracellular staining only).

    • i.
      Label a tube staining panel A extracellular antibody panel. Add 75 μL/sample flow cytometry buffer +10% overage. Add antibodies from materials and equipment Table 1.
  • c.
    Staining panel B (extracellular and intracellular stain).

    • i.
      Label a tube staining panel B extracellular antibody panel. Add 75 μL/sample flow cytometry buffer +10% overage. Add extracellular antibodies from materials and equipment Table 2.
    • ii.
      Label a tube staining panel B intracellular antibody panel. Add 75 μL/sample 1× BD Perm/Wash Buffer +10% overage. Add intracellular antibodies from materials and equipment Table 2.

Note: Antibodies should be diluted on the day of staining to minimize any non-specific interactions between antibody clones. Store at 4°C in the dark once dilutions have been prepared.

Note: In our experience, once reconstituted in DMSO live/dead fixable viability dye can be stored at -20°C with no decrease in performance after 2–3 freeze thaw cycles.

Table 1.

Antibody components of staining panel A

Antibody	Fluorochrome	Vendor	Clone	Staining compartment	Final dilution
CD16/CD32 Monoclonal Antibody (93)	N/A	ThermoFisher	N/A	extracellular	1:200
CD45.2	Alexa488	Biolegend	104	extracellular	1:400
CD45.1	BV605	Biolegend	A20	extracellular	1:400
CD3	BUV396	BD Biosciences	145-2C11	extracellular	1:200
CD4	BV711	BioLegend	RM4-5	extracellular	1:800
CD8	BV650	BioLegend	53-6-7	extracellular	1:800
CD19	BUV737	BD Biosciences	ID3	extracellular	1:800
TCR Vβ8.1, 8.2	PE	BioLegend	MR5-2	extracellular	1:200
ICOS	PE-Dazzle	BioLegend	C398.4A	extracellular	1:800
PD-1	BV421	BioLegend	29F.1.A12	extracellular	1:150

Table 2.

Antibody components of staining panel B

Antibody	Fluorochrome	Vendor	Clone	Staining compartment	Final dilution
CD16/CD32 Monoclonal Antibody (93)	N/A	ThermoFisher	N/A	extracellular	1:200
CD45.2	Alexa488	BioLegend	104	extracellular	1:400
CD45.1	BV605	BioLegend	A20	extracellular	1:400
GATA3	PE	BioLegend	16E10A23	intracellular	1:20
Mouse IgG2b, κ	PE	BioLegend	MPC-11	intracellular	1:1600

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD16/CD32 monoclonal antibody (93), 1:200 dilution	Thermo Fisher (Invitrogen)	14-0161-86
CD45.2, 1:400 dilution	Biolegend	109816
CD45.1, 1:400 dilution	Biolegend	110737
CD3, 1:200 dilution	BD Biosciences	563565
CD4, 1:800 dilution	BioLegend	100557
CD8, 1:800 dilution	BioLegend	100742
CD19, 1:800 dilution	BD Biosciences	564296
TCR Vβ8.1, 8.2, 1:200 dilution	BioLegend	140104
ICOS, 1:800 dilution	BioLegend	313531
PD-1, 1:150 dilution	BioLegend	135218
GATA3, 1:20 dilution	BioLegend	653804
Mouse IgG2b, κ, 1:1600 dilution	BioLegend	400311
Biological samples
Fetal calf serum (FCS)	MilliporeSigma	F4135
Bovine serum albumin (BSA)	Sigma-Aldrich	A9418
Dialyzed lyophilized collagenase type IV	Worthington Biochemicals	LS004210
Chemicals, peptides, and recombinant proteins
RPMI-1640 medium (Gibco)	Fisher Scientific	11875093
HEPES 1.0 M	Thermo Fisher Scientific	15630106
Dimethyl sulfoxide (DMSO) for molecular biology	Sigma-Aldrich	D8418
Sodium azide	Sigma-Aldrich	S2002
Phosphate buffered Saline (PBS)	Thermo Fisher Scientific	10010023
Ethylenediaminetetraacetic acid, (EDTA), 0.5 M Solution, molecular biology grade, ultrapure	Thermo Fisher Scientific	J15694.AE
Experimental models: Cell lines
PTCL lymphoma	Kuczynski et al.1	N/A
Critical commercial assays
LIVE/DEAD Fixable Blue Dead Cell Stain Kit	Thermo Fisher Scientific	L34961
Invitrogen™ eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set	Thermo Fisher Scientific	00-5523-00
1× RBC Lysis buffer	Thermo Fisher Scientific	00-4333-57
BD Cytofix Fixation buffer	BD Biosciences	554655
UltraComp eBeads™ Plus Compensation Beads	Thermo Fisher Scientific	01-3333-42
Experimental models: Organisms/strains
C57BL/6J mice, non-transgenic, 6–12 weeks of age, male or female	The Jackson Laboratory	000664
B6.SJL-Ptprca Pepcb/BoyJ mice, non-transgenic, 6–12 weeks of age, male or female	The Jackson Laboratory	002014
Software and algorithms
BD FACSDiva™ Software	BD Biosciences	N/A
FlowJo v10.8	TreeStar	N/A
Other
Corning® cell strainer (40 μm)	MilliporeSigma	CLS431750
Corning™ Falcon™ 50 mL Conical Centrifuge Tubes	Fisher Scientific	352070
Countess™ Cell Counting Chamber Slides	Thermo Fisher Scientific	C10315
Greiner CELLSTAR® 96 well plates (V-bottom clear wells)	MilliporeSigma	M9686
Pall AcroPrep 96-well 40 μm Filter plates	Thermo Fisher Scientific	17134601

CRITICAL: Follow all instructions on the safety data sheet when handling any reagents. Whilst reagents listed are generally safe to use under conventional BSL-1 laboratory conditions, institutional guidelines may vary.

Alternatives: Alternative vendors and clones may be considered, but should be titrated in control cells expressing the target of interest, to identify best performing concentrations (maximum signal-to-background) ahead of application to experimental samples.

Materials and equipment

The following tables show components for antibody staining cocktails.

Step-by-step method details

Liberation of immune cells from mouse spleen

Timing: 2–3 h (dependent on number of tissues evaluated)

The first step in the protocol is to isolate single cell suspensions containing T-cell cancer cells and host immune cells from murine spleen. This workflow can be readily adapted for other lymphoid tissues, including lymph nodes, thymus or bone marrow.

• 1.
  Dissect spleens into dissection medium kept on ice or at 18°C–23°C.

  Optional: If analysis of innate immune cell populations is required, perform collagenase digestion:

  • a.
    Transfer individual spleens into a well of a 6-well plate.
  • b.
    Add 4 mL of 18°C–23°C dissection medium.
  • c.
    Using a scalpel with a fresh blade, finely chop spleens into 15 or more pieces to maximize surface area for the following enzymatic digestion step.
  • d.
    Add 1 mL of 5–10 mg/mL Collagenase type IV, resulting in a final concentration of 1–2 mg/mL.
  • e.
    Incubate in a 37°C incubator (5% CO₂) for 25–30 min.
  • f.
    Stop reactions with addition of 2 mM EDTA.

    Note: If only T-cell and B-cell analysis is required from spleens, there is no need to perform collagenase digestion as these cells are liberated without the need for enzymatic digestion.

• 2.
  Pass spleens through a 40 μm cell strainer, placed on top of a 50 mL conical tube.

  • a.
    This process should be facilitated using the plunger of a 20 mL syringe to mash tissue through the mesh.
  • b.
    Rinse through with 2 mL ice cold flow cytometry buffer. Allow the cell strainer to drain and repeat 1–2 more times.
• 3.Pellet immune cells by centrifuging in a cold (4°C) benchtop centrifuge at 500 × g (approximately 1,200–1,500 RPM in a conventional lab benchtop centrifuge) for 5 min.

Optional: Red blood cell lysis can be performed at this stage, using 1× RBC Lysis buffer by following manufacturer’s instructions (https://assets.fishersci.com/TFS-Assets/LSG/manuals/00-4333.pdf). In our experience, an additional RBC lysis step is not necessary to facilitate downstream flow cytometry analysis, but it can aid in the accurate quantification of lymphoid cells.

• 4.Resuspend cell pellets in 2 mL chilled (4°C) flow cytometry buffer by gently vortexing.

Note: The aim of this step is to resuspend cells at a concentration of ∼2 × 10⁷ – 1 × 10⁸ cells/mL to facilitate downstream cell counting and sample aliquoting. It is anticipated that a spleen from an 8–12 week old female C57BL/6J mouse would comprise 5 × 10⁷-1 × 10⁸ cells. Resuspension volume should therefore be adjusted based on expected cell recoveries. Samples can be pelleted & re-suspended by following steps 3 and 4, or further diluted as needed.

• 5.Count cells using a hemocytometer or automated cell counter (e.g., Countess Cell Counting Chamber Slides and Countess 3 Automated Cell Counter (AMQAX2000)).

Note: In our experience, a counter stain such as trypan blue is not essential to accurately quantitate immune cells for the purposes of this protocol. Bright, circular immune cells with clearly defined membrane should be counted, and consistency in live cell counting should be maintained across samples.

• 6.Aliquot ∼2–5 × 10⁵ cells from each single cell suspension into a well of a 96-well v-bottom tissue culture plate (plate A) to be processed for staining panel A. The plate should be kept on ice or at 4°C for the further duration of the protocol.
• 7.Repeat step 6, to prepare a 96-well v-bottom tissue culture plate containing sample (plate B) for evaluation by staining panel B.

Note: This protocol can be readily adapted to evaluate immune cell populations from non-lymphoid tissues or solid tumors. In general, all non-lymphoid tissue preparation will involve an enzymatic digestion step with collagenase or a suitable alternative in order to liberate cells. Detailed digestion protocols for several tissues,⁵ or from solid tumors⁶ have been published.

Extracellular and intracellular flow cytometry staining

Timing: 3–5 h

The next step of the protocol involves staining of immune cell populations with fluorescently labeled antibodies to enable discrimination of cell populations.

• 8.Centrifuge plate A and plate B at 500 × g for 4 min in a 4°C benchtop centrifuge equipped with a microplate holder to pellet the cells. Remove the supernatant by flicking the plate into a sink following a single continuous motion [troubleshooting 1].

CRITICAL: Do not blot or dry residual liquid from the plate, to avoid dislodging cell pellets and/or cross-contamination between samples.

• 9.Resuspend cells in 100 μL live/dead blue stain, through gentle repetitive pipetting. Incubate at 4°C for 10–15 min.

Note: Use of a multichannel pipette can increase sample preparation consistency and reduce processing times.

• 10.Top up wells with 100 μL 4°C flow cytometry buffer and centrifuge (500 × g at 4°C) for 4 min. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.
• 11.Add 100 μL/well flow cytometry buffer and centrifuge (500 × g at 4°C) to pellet cells. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.
• 12.Resuspend cells from plate A in 75 μL/well staining panel A – extracellular antibody panel. Resuspend cells from plate B in 75 μL/well staining panel B – extracellular antibody panel. Perform surface staining for 30–60 min at 4°C in the dark [troubleshooting 2].

Note: Precise antibody cocktails can be modified as needed to address desired experimental questions. A broader list of antibodies and working dilutions that we have validated previously as part of our characterization of a T-cell lymphoma models have been published previously.¹

• 13.Top up wells in plate A with 125 μL 4°C PBS and centrifuge (500 × g at 4°C) for 4 min. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.
• 14.Resuspend plate A in 75 μL BD Cytofix Fixation buffer and incubate for 10–15 min at 4°C.
• 15.Top up wells in plate A with 125 μL ice cold PBS and centrifuge (500 × g at 4°C) for 4 min. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.

Note: BD Cytofix fixation buffer reagent contains paraformaldehyde which may be harmful. Use with care and dispose any waste according to institutional regulations.

• 16.Resuspend samples in plate A with 150 μL PBS.

Pause point: Plate A can be stored in the dark at 4°C for 24–48 h before analysis by flow cytometry.

• 17.Top up wells in plate B with 125 μL 4°C PBS and centrifuge (500 × g at 4°C) for 4 min. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.
• 18.Resuspend plate B in 75 μL/well fix/perm solution for 20–30 min at 4°C.
• 19.Top up wells in plate B with 125 μL ice cold Perm/wash buffer and centrifuge (500 × g at 4°C) for 4 min. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.
• 20.Resuspend plate B in 75 μL/well staining panel B – intracellular antibody panel. Incubate at 4°C in the dark for 30–45 min.
• 21.Top up wells in plate B with 125 μL ice cold Perm/wash buffer and centrifuge (500 × g at 4°C) for 4 min. Remove the supernatant through aspiration or by flicking the plate into a sink following a single continuous motion.
• 22.Resuspend samples in plate B with 75 μL perm/wash buffer. Top up with 75 μL PBS.

Note: fix/perm buffer contains formaldehyde, which is toxic. Use with care and dispose waste according to institutional regulations.

Note: fix/perm and perm/wash buffers contain a detergent which creates holes in the plasma and nuclear membranes, allowing fluorescent antibodies to penetrate and stain antigens. Cells must be kept in the presence of permeabilization reagent throughout all intracellular staining steps.

Pause point:plate B can be stored in the dark at 4°C for 24–48 h before analysis by flow cytometry.

Sample acquisition on 4 laser BD Fortessa flow cytometer with high throughput sample loader equipped

Timing: 2–4 h

The next step of the protocol involves acquisition of flow cytometry data. Fixed samples can be kept at 18°C–23°C for the duration of the protocol, and should be kept in the dark to prevent.

Optional: Samples can be passed through a 40 μm cell straining prior to acquisition. This can be done in a high-throughput manner using for example Pall AcroPrep 96-well 40 μm Filter plates. This will minimize the risk that any clumping of dead cells in the sample will clog the flow cytometer fluidics.

• 23.Prepare flow cytometry compensation controls using UltraComp eBeads™ Plus Compensation Beads according to manufacturer’s instructions (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2F01-2222.pdf).
• 24.The BD Fortessa should be calibrated and voltages optimized, acquisition parameters and plate labels should be set up using BD FACSDiva software all according to the instructions in the BD FACSDiva software reference manual.
• 25.
  Set up an acquisition worksheet in FACSDiva to enable visualization of sample acquisition.

  • a.
    Visualize dot plots showing a basic gating structure with total lymphocytes (FSC vs. SSC), live cells (viability dye-negative in FSC vs. viability dye), singlet cells (FSC-A vs. FSC-H) and congenic markers CD45.1 vs. CD45.2.
  • b.
    Set up plate on high-throughput screen (HTS) mode on the BD Fortessa.
  • c.
    Test sample flow rate using <5 μL spare sample and adjust if necessary to achieve a flow rate of 1,000–5,000 events/second.
  • d.
    Acquire and record data from all samples.
• 26.
  Following acquisition of samples, export data as an .FCS file.

  • a.
    Import files into FlowJo.
  • b.
    Using single color compensation controls, create and apply a compensation matrix and apply to experimental samples. For detailed instructions on creating and applying a compensation matrix, refer to the Traditional Compensation in FlowJo tutorial (https://www.youtube.com/watch?v=H17WUe7Vlbg, FlowJo Media).
  • c.
    Analyze data in FlowJo software.

CRITICAL: Antibody fluorophores must be compatible for use with the particular flow cytometer instrument configuration and laser set up. Alternative antibody panels and/or differential use of fluorophores can be optimized and/or considered, depending on the flow cytometry capabilities available.

Expected outcomes

Successful completion of the protocol should enable analysis of host and transferred cell populations, based on gating of congenic markers. An example of the application of this staining panel to C57BL/6J mouse splenocytes (CD45.2+, with no transferred tumor cells) is shown in (Figure 1). Using FSC-A and SSC-A parameters, and gating on live/dead UV-450 negative populations, live lymphocytes can be distinguished. Further gating using FSC-H and FSC-A, followed by SSC-H and SSC-A parameters enables distinction of singlet cells, and excludes any doublet cell populations that are undesirable for single-cell analysis. CD45.2+ cells comprise the vast majority of this population, with a fractional population of CD45.2-CD45.1- seen, and likely comprising non-hematopoetic cells.

Figure 1.

C57BL/6J (CD45.2) mouse splenocytes stained with stainingpanel A

Dot plots of cells acquired from a normal mouse spleen obtained from a tumor-free C57BL/6J mouse. Lymphocytes negative for viability dye were gated for singlets and subsequently CD45.2+ CD3+ T-cells. Normal T-cells demonstrate a mix of predominantly CD4+ or CD8+ T-cells. A minority of T-cells are positive for TCRvβ8 and few expression activation and exhaustion markers ICOS and PD-1, respectively.

Further subsetting of CD45.2+ cells reveals populations of CD19+ B-cells, CD3+ T-cells. CD3+ T-cells can be further gated to reveal CD4+ T-helper and CD8+ T-cytotoxic populations. The vast majority of T-cells in 6–12 week old C57BL/6J mice housed in conventional specific-pathogen free conditions are naïve, typically lacking high expression of PD-1 and ICOS surface markers that are indicative of activated or exhausted T-cells (e.g., following recognition of tumor antigens).^7,8,9 A polyclonal T-cell repertoire is typified by a breadth of TCR Vβ usage, and inclusion of antibodies recognizing TCRvβ8 reveals incorporation into 19.8% of T-cell receptors¹ (Figure 1).

Application of staining panel A to B6.SJL-Ptprca Pepcb/BoyJ (CD45.1+) mouse strain harboring transplanted T-cell lymphomas (CD45.2) reveals differential cellular composition (Figure 2). CD45.1+ host cells can be identified, alongside a larger population of CD45.2+ tumor cells. In our model, host cells were largely devoid of T-cells but contained abundant B-cells (CD19+), which may have resulted from out-competition of host T-cells by the lymphoma for homeostatic cytokines. In contrast, tumor cells present as CD3hi-med indicative of their T-cell origin. They are broadly CD4+, with some evidence of receptor downregulation¹ In contrast to host naïve T-cells, lymphomas express high levels of PD-1 and ICOS, which may confer cells with immunomodulatory properties. Finally, lymphomas are synchronously TCRvβ8+, indicative of their clonal origin. Thus staining panel A has successfully enabled identification of host and lymphoma tumor cells from a single splenocyte preparation, and informs on differential phenotypes that may have mechanistic relevance.

Figure 2.

Splenocytes from a B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mouse that was engrafted with CD45.2+ congenic lymphoma cells, analyzed with staining panel A

CD45.2+ transplantable cells expressed CD3 and heterogenous levels of CD4, but were absent for CD8. In contrast with normal T-cells in (Figure 1), CD45.2+ highly expressed ICOS, PD-1 and TCRvβ8, suggesting a clonal activated T-cell expanded in the mouse. CD45.1+ cells representing residual host immune cells largely comprised CD19+ B-cells.

Evaluation of intracellular biomarkers represents a further important method to characterize lymphoma cells. Staining panel B was similarly applied to evaluate expression of the GATA3 transcription factor in splenocytes from healthy C57BL/6J (CD45.2) mice or B6.SJL-Ptprca Pepcb/BoyJ (CD45.1+) mice harboring T-cell lymphomas (Figure 3). GATA3 is a lineage marker associated with activated Th2 polarized cells (including T-helper and T-follicular helper).^10,11 Analysis revealed negligible expression of GATA3 in healthy C57BL/6J mice, or in the CD45.1+ host cell compartment of lymphoma bearing B6.SJL-Ptprca Pepcb/BoyJ mice. However, we observed elevated GATA3 expression in lymphoma cells. This data informed in the tumorigenesis process by supporting a Th2/Tfh cell-of-origin hypothesis.¹

Figure 3.

Splenocytes from a B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mouse was engrafted with CD45.2+ congenic lymphoma cells, analyzed with staining panel B

Overlaid dot plots and histograms demonstrate a positive-shift of GATA3 staining in CD45.2+ cells (T-cell lymphoma) vs. isotype control. CD45.2+ cells in the normal mouse spleen and CD45.1 host cells do not express GATA3.

Collectively, these methods enable new insights into transplantable lymphoma tumor models and may be applied to other syngeneic leukemia or lymphoma scenarios. Moreover, utility of staining panels containing CD45.1 and CD45.2 congenic markers can also be applied support additional lymphocyte transfer studies.³

Limitations

The protocols herein rely on the continued expression of congenic markers on tumor cells in order to distinguish them from host cells. Tumorigenesis may result in changes or downregulation of conventional surface receptors, which may be endogenous to the model or may occur over time. When applying these protocols to additional tumor cell populations, loss of CD45 surface expression would make identification of lymphoma cells challenging with this protocol, and has been reported in certain hematopoietic malignancies.¹² Whilst gating on host CD45+ cells could represent an alternative approach, there is a risk of overlap between host and tumor cells if a single parameter is used. Identification of an alternative tumor-specific marker can be considered to circumvent this challenge. Alternatively, use of a membrane dye such as CFSE can be utilized to fluorescently label cell membranes and facilitate evaluation of homeostatic kinetics.¹³ However, membrane dyes will dilute upon cell division, and thus this method would be most suitable to short-term kinetic studies of slowly proliferating cancers.

A second limitation is the requirement for multiple fluorescent antibodies to be incorporated into the staining panel for the purpose of identifying and distinguishing host and lymphoma cells. This necessitates development and application of a larger cell panel if additional cellular characterization is required. This limitation can be circumvented through the use of multiple staining panels, and/or is mitigated by analysis on flow cytometers that are equipped for analysis of high numbers of fluorescent parameters. We anticipate this limitation will be negated with the continued evolution of multiparametric flow cytometry technology.

Finally, it should be considered that the application of these protocols to novel or uncharacterized hematopoietic cancer cells requires extensive method development, control experiments and evaluation to enable rigorous conclusions to be drawn. Many of the control experiments have been published alongside our initial characterization of the model, alongside reviewers’ questions and our responses.¹ Whilst this publication represents a further guide for novel model and method development, distinctive models are likely to require nuanced tuning of the experimental approaches.

Troubleshooting

Problem 1

Spillover between wells when staining in plates.

As indicated in step 8, the protocol necessitates multiple washing and staining steps in a 96-well plate. There is potential for contamination between wells if the supernatant if care is not taken, and particularly when utilizing the plate flicking technique to remove supernatant. It is important not to blot the plate dry following a wash, as this can disrupt cell pellets leading to sample loss. Video instruction of the plate flicking method to remove supernatant is available at the following link: https://www.youtube.com/watch?v=5BLAQ117mCo.

Potential solution

An alternative to the plate flicking technique to remove supernatant during washes is to employ careful aspiration with a multichannel pipette. If cross-well contamination is still of concern, spacing of samples with a blank well in between can be considered. Staining can also be performed direction in 5 mL round bottom polystyrene tubes (Corning Falcon, Catalog number 352052).

Problem 2

Optimization of staining concentrations.

As indicated in step 12, there is potential need to optimize concentrations of antibodies in order to facilitate optimal staining and resolution of cell populations. Typically, antibody staining is performed with saturating concentrations of surface antibodies and flow cytometer voltage settings are adjusted accordingly. Antibody dilutions used in this study are depicted in Tables 1 and 2, but may need to be optimized.

Two underlying scenarios are envisaged. Firstly, staining concentrations may need to be increased, in situations where higher levels of antibody target lead to depletion of fluorescent antibodies in solution. This scenario can apply if higher concentrations of cells are being stained in a given sample, or if lymphoma cells express a particularly high expression level of an antibody target. Secondly, staining concentrations may need to be decreased, for example if staining is too bright for a particular flow cytometric detector and if changing the machine voltage is undesirable due to effects on multiparametric compensation parameters.

Potential solution

In order to optimize staining, representative single cell suspensions should be stained with a serial dilution of an antibody. Evaluation of GeoMean fluorescent intensity versus concentration on a curve will identify Emax concentration. In our experience, the staining panels described are sufficiently robust at the recommended staining concentrations, and thus should provide a good starting point. Thus optimization of a particular fluorescent antibody stain is only recommended if issues are uncovered during pilot experiments, or if alternative antibodies are being incorporated into the staining panels, or if there are other significant deviations in the methods or equipment described. For stains that do not clearly separate positive from negative populations, include FMO (fluorescence minus one) controls to define positive gate positions.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the techical contact Elizabeth Kuczynski, Elizabeth.kuczynski@astrazeneca.com, or the lead contact Charles Sinclair, csinclair@flagshippioneering.com.

Materials availability

No newly generated materials are associated with this protocol.

Data and code availability

There are restrictions to the availability of the raw flow cytometry dataset analyzed in this manuscript, which is proprietary to AstraZeneca. Dataset requests can be made by contacting the lead author EAK.