Protocol to co-culture SCLC cells with human CD8⁺ T cells to measure tumor cell killing and T cell activation

Summary

Small-cell lung cancer (SCLC) is an aggressive malignancy with immunosuppressive tumor microenvironment limiting immunotherapy efficacy. Here, we present a protocol to assess T cell activation and the ability of lurbinectedin to enhance anti-tumor responses using in vitro co-cultures of SCLC cells and human CD8 T cells. We describe steps for cell seeding, treatment, co-culture setup, and assessing cell viability. This protocol provides a platform to study anti-tumor T cell responses and develop new SCLC therapies.

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

Graphical abstract

Highlights

• •Co-culture of SCLC cells with HLA-matched human CD8 T cells
• •Steps to evaluate T cell activation and tumor cell viability
• •Details to quantify lurbinectedin-induced cytokine production via ELISA
• •Platform to interrogate human anti-tumor effector T cell responses in SCLC

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

Small-cell lung cancer (SCLC) is an aggressive malignancy with immunosuppressive tumor microenvironment limiting immunotherapy efficacy. Here, we present a protocol to assess T cell activation and the ability of lurbinectedin to enhance anti-tumor responses using in vitro co-cultures of SCLC cells and human CD8 T cells. We describe steps for cell seeding, treatment, co-culture setup, and assessing cell viability. This protocol provides a platform to study anti-tumor T cell responses and develop new SCLC therapies.

Before you begin

Timing: 7 days

Immune cold nature of SCLC, the most aggressive type of lung cancer provides limited scope with immunotherapy.^2,3 In this protocol, we used co-cultures of SCLC cells and human CD8 T cells in-vitro and assessed the activation of T cells as well as the efficiency of lurbinectedin to enhance anti-tumor responses.^4,5 We use HLA-A02:01-expressing SCLC cells pulsed with NY-ESO-1 peptide and CD8+ T cells with NY-ESO-1-specific T cell receptors to estimate T cell-mediated tumor killing and IFNγ production using ELISA.^1,6,7,8 The protocol described here assumes that the user is familiar with and has access to standard enzyme linked immunosorbent assays (ELISA), flow cytometry and plate reader. We provide detailed description of the steps to measure T cell reactivity and tumor cell death in the co-cultures. While our protocol utilizes commercially available NY-ESO-1-specific T cells from Charles River, NY-ESO-1 reactive T cells can be also generated by adapting the protocol described by Bozkus et al.⁹ and using HLA-02:01-positive PBMC (commercially available through Cytologicsbio). Additionally, human T cells can be engineered to express the NY-ESO-1-specific TCR (clone 1G4).^10,11

It is advisable to confirm surface major histocompatibility complex class I (MHCI) expression in the SCLC tumor lines by flow cytometry prior to the beginning of the experiment. If necessary, SCLC tumor line treatment with recombinant human IFNγ will augment surface MHCI expression.

All reagents are kept sterile following standard aseptic techniques. Cells cultures are maintained in a humidified incubator at 37°C with 5% CO₂.

Cell culture:

• 1.Maintain suspension SCLC cells in complete RPMI media.
• 2.Add fresh media on top of depleted media every other day.
• 3.Once the growth looks confluent, take out the cell-containing media.
• 4.Centrifuge the media containing cells at 500×g for 3 min at 25°C.
• 5.Prepare single cell suspension using 0.25% Trypsin-EDTA for 1 min on the cell pellet.
• 6.Neutralize the cells with 4 mL of complete RPMI media (4 volumes relative to trypsin).
• 7.Ensure a homogeneous cell suspension by pipetting.
• 8.Determine cell number using a 1:1 (10 μL:10 μL) mixture of cell suspension and 0.4% trypan blue.
• 9.Split the cells into 1:3 flasks when the confluency reaches 0.5 × 10⁶ cell/mL.

Note: Test and authenticate SCLC cell lines by short tandem repeat profiling (DNA fingerprinting) and routinely test for mycoplasma species before any experiments. Use the cells within the first three to five passages for any experiments. Perform cell counts before seeding for any assay and use cells only if viability is >75%.

Media preparation

Timing: 10 min

• 10.Prepare complete RPMI media as described in the materials and equipment section.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
α-PD-L1 antibody (atezolizumab) (dilution 1:500)	MedChemExpress (MPDL3280A)	HY-P9904
Ultra-LEAF Purified anti-human HLA-A,B,C antibody or α-MHC-I (dilution 1:112)	BioLegend	Clone: W6/32, Cat# 311428, RRID: AB_2561492
ImmunoCult human CD3/CD28/CD2 T cell activator (dilution 1:100)	STEMCELL Technologies	10990
Chemicals, peptides, and recombinant proteins
NY-ESO-1 peptide (SLLMWITQC)	JPT	SP-MHCI-0001-2
Lurbinectedin	Selleckchem	Cat #S9603
Critical commercial assays
CellTiter-Glo Assay	Promega	G7571
CD8+ T Cell Isolation Kit, human	Miltenyi Biotec	130-096-495
ELISA MAX Deluxe Set Human IFN-γ	BioLegend	430104
ELISA MAX Deluxe Set Human CXCL10	BioLegend	439904
Experimental models: Cell lines
Anti-NY-ESO-1 T cells	Charles River	ASCT-1093
H69	ATCC	RRID: CVCL_1579
Software and algorithms
GraphPad Prism Ver. 9.0	GraphPad Software, Inc., San Diego, CA, USA	RRID: SCR_002798
Other
PBS	Corning	21-040-CV
RPMI 1640 media	Corning	10-040-CV
Fetal bovine serum heat inactivated	Sigma-Aldrich	F4135
Penicillin-streptomycin	Gibco	15140-122
MEM NEAA	Gibco	11140-050
Trypan blue stain	Invitrogen	T10282
96-well flat-bottom opaque plate	Thermo Fisher Scientific	165305
96-well, cell culture-treated, U-shaped-bottom microplate	Corning	3799
Sodium pyruvate	Gibco	11360-070

Materials and equipment

Complete RPMI media

Reagent	Final concentration	Amount
RPMI 1640 media	N/A	174 mL
Fetal bovine serum	10%	20 mL
Penicillin-streptomycin	1%	2 mL
Non-essential amino acids (MEM NEAA)	1x	2 mL of 100X stock
Sodium pyruvate	1X	2 mL of 100X stock
Total	N/A	200 mL

Note: Fetal bovine serum, L-Glutamine and penicillin-streptomycin aliquots are stored at −20°C and thawed in a 37°C water bath prior to use. RPMI media, NEAA and sodium pyruvate are stored at 4°C. After mixing all reagents, complete RPMI media solution can be stored at 4°C for up to 4 weeks.

NY-ESO-1 peptide stock solution

Reagent	Final concentration	Amount
NY-ESO-1 peptide	N/A	1 mg
DMSO	N/A	1 mL
Total	1 mg/mL	1 mL

Note: Aliquot and store NY-ESO-1 peptide solution at −20°C for up to 6 months.

Working solutions for 2x NY-ESO-1 peptide (20 wells total)

Reagent	Final concentration	Amount
NY-ESO-1 peptide stock solution	1 mg/mL	10 μL
Complete RPMI media	N/A	990 μL
Total	10 μg/mL	1 mL

Note: NY-ESO-1 peptide stock solution is stored at −20°C and thawed right before use. 2x working solution should be prepared fresh for every use.

2x α-PD-L1 antibody solution (for 10 wells total)

Reagent	Final concentration	Amount
α-PD-L1 antibody stock solution	60 mg/mL	2 μL
Complete RPMI media	N/A	998 μL
Total	120 μg/mL	1 mL

Note: α-PD-L1 antibody stock solution is stored at −80°C. Thaw aliquots right before use on ice. Prepare fresh working solution before every use.

2x α-human HLA-A, B, C antibody solution (for 10 wells total)

Reagent	Final concentration	Amount
α-human HLA-A, B, C antibody stock solution	2.24 mg/mL	8.93 μL
Complete RPMI media	N/A	991.07 μL
Total	20 μg/mL	1 mL

Note: α-human HLA-A, B, C antibody stock solution is stored at 4°C. Prepare fresh 2x working solution before every use.

Step-by-step method details

Seed SCLC cell lines

Timing: 30 min

The following steps were performed on Day 0 to prepare and seed SCLC H69 cells prior to treatment with lurbinectedin.

• 1.Transfer H69 SCLC suspension culture (RRID# CVCL_1579) into 15 mL centrifuge tubes.
• 2.Pellet the cells by centrifugation at 500xg for 3 min at 25°C.
• 3.Discard the supernatant, and re-suspend the cells in 1X sterile DPBS. Centrifuge the suspension again at 500xg for 3 min at 25°C.
• 4.Discard the DPBS supernatant, and re-suspend the cell pellet in 1 mL of 0.25% trypsin-EDTA solution for 1 min to dissociate clumps and achieve a single-cell suspension.
• 5.Neutralize the trypsinized cells with 4 mL of complete RPMI media (4 volumes relative to trypsin), ensuring a homogeneous cell suspension.
• 6.Determine the number of cells using a 1:1 mixture of cell suspension and 0.4% trypan blue stain, using a cell counter.
• 7.After counting, seed 1 mL of 10⁶ cells per mL into each well of a 6-well tissue culture dish in complete RPMI media.

Note: SCLC cells often grow in aggregates which may result in inconstancy among the replicates during readout. Please refer to troubleshooting section (problem 1) for address.

Thaw T cells

Timing: 45 min

The following steps are performed on day 1 and describe the protocol to start peptide-reactive T cell cultures from cryopreserved vials. The cells are allowed to rest overnight before using them in co-culture assays.

• 8.Warm complete RPMI media in a 37°C water bath for 5–7 min to bring it to 25°C.
• 9.Transfer the cryovials containing cryopreserved NY-ESO-1-specific T cells directly from liquid nitrogen storage into 37°C water bath.
• 10.As soon as the cells are thawed with a small ice pellet remaining, remove the cryovial from the water bath.
• 11.Add 9 mL complete RPMI media to a 15 mL falcon tube.
• 12.Transfer 1 mL of the cryopreserved cells in drop-wise manner to the 15 mL Falcon tube with 9 mL media and mix gently by pipetting.
• 13.Centrifuge the cells at 400xg for 4 min.
• 14.
  Aspirate the supernatant and re-suspend the cells in 2 mL of fresh media for a second wash.

  • a.
    Re-suspend cells in complete RPMI media and take an aliquot for cell counting.
  • b.
    Count the total number of cells.
  • c.
    Centrifuge the remaining cells at 400xg for 4 min.
  • d.
    Aspirate supernatant.

Note: We routinely re-suspend cells in 1–5 mL complete RPMI media depending on cell density. To count, we make 1:1 ratio of cell suspension and 0.4% trypan blue stain and use 10 μL for counting cell number using a hemocytometer.

• 15.Re-suspend the T cells at 10⁶ cells/mL with complete RPMI media.
• 16.Seed cells at 10⁶ cells/well in 1 mL media in a 24-well plate.

Note: If the cell count is 3–5 million, seed the cells in one well of a 6-well plate at 1-2 x 10⁶ cells/mL. If the cell count is > 5 million, transfer the cells to a T25 flask at 1-2 x 10⁶ cells/mL.

• 17.Culture cells overnight in a humidified incubator at 37°C with 5% CO₂.

Treat SCLC cell lines with vehicle/lurbinectedin

Timing: 30 min

The following steps are performed on Days 1 to pre-treat 2D SCLC cell lines with lurbinectedin for 24 h before initiating the co-culture assay.

• 18.On Day 1, 24 h after SCLC tumor cell line seeding, treat three wells (each containing 1 × 10⁶ cells) with 10 nM lurbinectedin.
• 19.Treat additional three wells with DMSO using a volume equivalent to that used for the lurbinectedin treatment.

Note: Lurbinectedin concentration was selected based on tumor cell viability¹² and immune-modulatory effect of lurbinectedin.⁸

Co-culture assay

Timing: 4 h

The co-culture assay is set up on day 2, after 24 h pre-treatment of SCLC cells with lurbinectedin. First, SCLC cell lines are pulsed with NY-ESO-1 peptide for 2 h at 37°C. Then, excess unbound peptide is washed off and NY-ESO-1-specific T cells are added to the culture. Finally, any drugs or blocking antibodies, such as α-PD-L1 or α-MHC-I are added to the culture. The co-culture is performed for 20 h.

• 20.Warm complete RPMI media in a 37°C water bath for 5–7 min to bring it to 25°C.
• 21.
  Calculate the number of required wells for co-culture assay (Figure 1).

  • a.
    Each condition is performed in triplicate wells.
  • b.
    Assay conditions:

    • i.
      2D SCLC cell line treated with vehicle or lurbinectedin + NY-ESO-1 peptide + T cells.
    • ii.
      2D SCLC cell line treated with vehicle or lurbinectedin + NY-ESO-1 peptide + T cells + α-MHC-I antibody.
    • iii.
      2D SCLC cell line treated with vehicle or lurbinectedin + NY-ESO-1 peptide + T cells + α-PD-L1 antibody (Atezolizumab).
  • c.
    Include the following control wells:

    • i.
      2D SCLC cell line treated with vehicle or lurbinectedin only (without T cells or NY-ESO-1 peptide).
    • ii.
      T cells only (without cancer cells or NY-ESO-1 peptide).
    • iii.
      2D SCLC cell line treated with vehicle or lurbinectedin + T cells (without NY-ESO-1 peptide).
    • iv.
      T cells stimulated with Immunocult to demonstrate polyclonal T cell activation.

Note: We routinely stimulate 0.1 to 0.5 million T cells with 1–2 μL of Immunocult human CD3/CD28/CD2 T cell activator to serve as positive control.

Note: Please refer to troubleshooting section (problem 2) for addressing insufficient cell numbers to test all desired conditions.

• 22.
  Re-suspend the vehicle and lurbinectedin pre-treated SCLC cells at 10⁶ cell/mL.

  • a.
    Transfer the vehicle- and 10 nM lurbinectedin-treated H69 cells into individual microcentrifuge tubes.
  • b.
    Centrifuge the cells at 500xg for 3 min at 25°C to remove the drug and DMSO.
  • c.
    Discard the supernatant and re-suspend the cells in 10 mL of 1X sterile DPBS. Centrifuge again at 500xg for 3 min at 25°C.
  • d.
    Discard the DPBS supernatant, and re-suspend the cell pellet in 1 mL of 0.25% trypsin-EDTA solution for 1 min to generate single cell suspension.
  • e.
    Neutralize the trypsinized cells with 4 mL complete RPMI media (4 volumes of Trypsin) and mixed well to generate a homogeneous cell suspension.
  • f.
    Next, count the number of cells using 1:1 ratio of cell suspension and 0.4% trypan blue stain using a cell counter.
  • g.
    After enumeration take 1 × 10⁶ cells into fresh micro-centrifuge tubes separately for each replicate of vehicle and drug treatment.
  • h.
    Centrifuge the cells at 500xg for 3 min at 25°C to remove the drug and DMSO.
  • i.
    Discard the media supernatant containing drug or DMSO, and re-suspend the cells in complete RPMI media.
• 23.
  Add 50 μL of vehicle or lurbinectedin treated SCLC cell lines to the respective wells of a 96-well U-bottom plate to seed 50,000 cells/well.

  • a.
    Add 50 μL media to the remaining wells.
• 24.
  Add NY-ESO-1 peptide to the tumor cell lines in the respective wells of the 96-well U-bottom plate.

  • a.
    Prepare 2x NY-ESO-1 peptide working solution in complete RPMI media.
  • b.
    Add 50 μL of the 2x solution to the respective wells yielding a final concentration of 5 μg/mL.
  • c.
    Add 50 μL media to the remaining wells.
  • d.
    Mix well by gentle pipetting.
• 25.Incubate the plate in a humidified incubator at 37°C with 5% CO₂ for 2 h.
• 26.Following the 2 h peptide pulse, make up the final volume in all wells to 200 μL by adding 100 μL of fresh media.
• 27.Centrifuge at 400xg for 4 min.
• 28.Carefully remove 150 μL of the supernatant with a multichannel pipette without disturbing the cell pellet.
• 29.
  Do a second wash by adding 150 μL of fresh media.

  • a.
    Centrifuge at 400xg for 4 min.
  • b.
    Carefully remove 150 μL of the supernatant without disturbing the cell pellet.
  • c.
    Remaining volume in the well should be 50 μL.
• 30.
  Prepare NY-ESO-1-specific T cells at 10⁶ cells/mL in complete RPMI media.

  • a.
    Collect the T cells into a 15 mL falcon tube.
  • b.
    Make up the volume to 15 mL using PBS.
  • c.
    Centrifuge at 400xg for 4 min.
  • d.
    Aspirate the supernatant and re-suspend in 3 mL PBS.
  • e.
    Take an aliquot for cell counting and centrifuge the remaining cells at 400xg for 4 min for a second wash.
  • f.
    Count the total number of cells.
  • g.
    Aspirate the supernatant and re-suspend at 10⁶ cells/mL in complete RPMI media.
• 31.
  Add 50 μL of NY-ESO-1-specific T cells to the respective wells of the plate to seed 50,000 T cells/well.

  • a.
    Add 50 μL media to the remaining wells.
  • b.
    Total volume in each well should be 100 μL.
• 32.
  Finally, add 100 μL of blocking antibodies such as α-PD-L1 or α-MHC-I at 2x concentration.

  • a.
    Prepare 2x α-PD-L1 and α-MHC-I antibodies in complete RPMI media.
  • b.
    Add 100 μL of the 2x solutions to the respective wells to obtain final concentrations of 60 μg/mL α-PD-L1 and 10 μg/mL α-MHC-I antibody.
  • c.
    Add 100 μL of media to the remaining wells.
• 33.
  Mix all wells with a multichannel pipette.

  • a.
    Final volume in each well is 200 μL.
• 34.Perform the co-culture assay for 20 h by incubating the plate in a humidified incubator at 37°C with 5% CO₂.

Figure 1.

Plate map of the different co-culture conditions

96 well plate depicting the different conditions and plate set up for the co-culture experiment.

Harvest the supernatants for ELISA

Timing: 15 min

This is the protocol for collecting supernatants following 20 h of co-culture on day 3. The cytokines released in these supernatants can then be analyzed using commercially available ELISA kits. The following steps can be performed under non-sterile conditions.

• 35.After 20 h of co-culture, remove the plate from the humidified incubator.
• 36.Centrifuge the plate at 400xg for 4 min.
• 37.
  Using a multichannel pipette, collect 150 μL of supernatants into a new 96-well U-bottom plate.

  • a.
    The supernatants can be stored at −20°C for up to 1 year.
  • b.
    Avoid freeze-thaw cycles.
• 38.Analyze cytokine production (such as IFNγ, CXCL10, etc.) by ELISA (according to the manufacturer’s protocol).

Viability assay

Timing: 1 h

The following steps describe the protocol for assessing the total number of viable cells after 20 h of co-culture on day 3 using Promega’s CellTiter-Glo assay. These steps can be performed under non-sterile conditions.

• 39.Warm complete RPMI media in a 37°C water bath for 5–7 min to bring it to 25°C.
• 40.After harvesting supernatants, add 50 μL of fresh media to all the wells to yield a final volume of 100 μL in each well.
• 41.
  Prepare the CellTiter-Glo working reagent according to manufacturer’s instructions (Promega CellTiter-Glo luminescent cell viability assay).

  • a.
    Thaw the buffer of cellTiter-Glo reagent at 25°C prior to use, while keeping the substrate (powder in the amber color bottle) at 4°C, protected from light.
  • b.
    Once thawed, transfer the appropriate volume (10 mL for Cat.#G7570 and G7571; 100 mL for Cat.#G7572 and G7573) of CellTiter-Glo Buffer into the amber bottle containing CellTiter-Glo substrate to reconstitute the lyophilized enzyme/substrate mixture. This forms CellTiter-Glo Reagent.
  • c.
    Mix by gently vortexing, swirling or inverting the contents to obtain a homogeneous solution.
• 42.Equilibrate the CellTiter-Glo reagent and the cell culture plate at 25°C for 30 min.
• 43.
  Add 100 μL of the CellTiter-Glo reagent to all the wells using a multichannel pipette to obtain a 1:1 mixture of cells/media and CellTiter-Glo reagent.

  • a.
    Mix well by pipetting to induce cell lysis.
• 44.Incubate the plate at 25°C for 10 min.
• 45.Transfer the contents from all the wells into a 96-well flat bottom opaque-plate.
• 46.Record luminescence reading using an appropriate instrument.
• 47.
  Use software such as Microsoft Excel or GraphPad Prism to analyze the data.

  • a.
    Directly plot the luminescence values as bar graphs and normalize to the no peptide control condition.

Note: Luminescence reading gives the total number of viable cells in each well. The data analysis can be done by plotting the luminescence values from the plate reader on Prism directly. Use the "no peptide condition" as control and normalize all luminescence values by dividing by the reading of "no peptide condition" and plot that as bar graphs.

ELISA: Cytokine excretion of lurbinectedin-treated SCLC cell lines

Timing: 24 h

• 48.Follow the manufacturer’s (Bio Legend) protocol for ELISA to detect secreted levels of interferons and/or cytokines (Kit details mentioned in the key resources table).

Note: All washes should be performed similarly without allowing the well to completely dry. Samples should be diluted in 1X Assay Diluent A before adding to the wells. Seal plate and incubate at room temperature for 2 h on countertop. If the samples were diluted, multiply by the appropriate dilution factor. It is critical that the sample analyte absorbance should fall in the mid-range of the standard curve absorbance value, if not test samples need to be reanalyzed at a higher or lower dilution as appropriate. Please refer to troubleshooting section (problems 3, 4, and 5) for addressing low and variable concentration of secreted analyte.

Expected outcomes

We describe a protocol to assess T cell activation and killing of cancer cells in response to small molecule inhibitor using a co-culture to determine cell viability and cytokine production. These are simple and robust assays that measure T cell activation even when the peptide-reactive T cell population is limited. We have successfully performed this assay using only 1.1 million NY-ESO-1-specific CD8 T cells. This protocol is applicable to other HLA-02:01-positive cancer models and can be easily adapted for HLA-02:01-presented tumor-antigen-derived peptides to assess peptide-specific T cell activation. While we present readout as cell viability (in Figure 2 of our associated paper⁸) but ELISA to detect IFNγ production can be another readout, these protocols can be adapted to determine the production of different cytokines via ELISA, ELISPOT or intracellular cytokine staining.

Figure 2.

Determination of secreted level CXCL10 upon lurbinectedin treatment

(A) Bar graphs showing variability and low secreted level of cytokine CXCL10 upon 16 and 24 h post-10 nM lurbinectedin treatment compared to vehicle treatment in DMS114 cells when the cell free media was not concentrated. The data represents the means ± SEM of 3 biological replicates (n = 3), one-way ANOVA was performed followed by paired two-tailed Student’s t-test to compare between two groups. ns, no significance; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

(B) Bar graphs showing low variability and significant increase in secreted level of cytokine CXCL10 upon 16 and 24 h post-10 nM lurbinectedin treatment compared to vehicle treatment in DMS114 cells upon concentration of cell free media with Pierce Protein Concentrators. The data represent the means ± SEM of 3 biological replicates (n = 3), one-way ANOVA was performed followed by paired two-tailed Student’s t-test to compare between two groups. ns, no significance; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Quantification and statistical analysis

Data are expressed as means ± standard error of mean (SEM). Perform one-way ANOVA or the Dunnett’s test for multiple group comparisons followed by Student’s t-test (to compare between two groups in a multiple group cohort) using GraphPad Prism to determine statistical significance among the biologically distinct groups. p values of students’ t-test less than 0.05 considered to be statistically significant (ns > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Data expressed as the mean ± SEM.

Limitations

This assay measures the total number of viable cells after 20 h co-culture of T cells with cancer cell lines. One of the limitations of the assay is that it is not able to distinguish if these live cells are T cells or cancer cells, and if there is a reduction in viability of cancer cells upon co-culture. To address this, cancer cells and T cells can be labeled with different fluorescent dye such as Cell Trace or CFSE, or engineered to express fluorescent proteins such as GFP, RFP, etc. Tumor cell death can be evaluated by fluorescence reader, flow cytometry, microscopy or IncuCyte.

T cell activation in this assay is measured by cytokine production following co-culture with cancer cells since we were limited by the number of NY-ESO-1-specific CD8 T cells. If peptide-specific T cell numbers are not a limiting factor, then another way to assess direct T cell activation is by performing flow cytometry for activation induced markers (CD25, CD137, CD134, etc), cytokines (IFN-γ, GM-CSF, TNFα, etc), cytolytic molecule production (CD107a, Granzyme A/B) following 16 h co-culture in the presence of protein transport inhibitors.^13,14,15

It is important to consider the MHC-restriction of commercially available peptide-reactive T cells and select cancer cells that express the same MHC alleles. To overcome the requirement of cognate TCR peptide/MHC interaction, co-cultures can be performed in the presence of Staphylococcal enterotoxin B super-antigen as we have previously shown.¹⁶ Since SEB engages a subset of TCRβ chains, the proportion of activated T cells will be donor dependent. Additionally, not all tumor cell lines express MHC class II. In some tumor lines, MHC class II can be induced upon overnight stimulation with IFNγ.

Troubleshooting

Problem 1

Variability among the replicates. SCLC cells often grow in aggregates and forms clumps, which result in irregular distribution while cell seeding. This may result in higher variations among the replicates.

Potential solution

Re-suspend the cells very well while neutralizing trypsin before seeding. Gently pipette the media containing the cells up and down multiple times to make sure single cell suspension.

Problem 2

Inadequate number of peptide-specific T cells to test all assay conditions.

Potential solution

If T cells are limited in numbers, you may reduce control wells to duplicates instead of triplicates. You may also omit T cells only (without cancer cells or peptide) control wells to be able to include all desired assay conditions. Additionally, the user may consider generating NY-ESO-1 T cell lines by stable transduction of NY-ESO-1-specific TCR¹⁷ or derivation of NY-ESO-1 T cell lines as previously reported.⁹

Problem 3

Low level of cytokines in the sample. SCLC cells treated with drug and vehicle may be too diluted for effective detection of cytokines and chemokines using the ELISA protocol.

Potential solution

Centrifuge the cell-free media at 2500 x g for 3 min following drug treatment on the cultured cells. Then add the collected supernatant up to 2–6 mL into a pre-cleaned concentrator tube (Pierce Protein Concentrators, PES, Thermo Fisher Scientific, Catalog number: 88514) and centrifuge at 4000 x g until a ∼10-fold concentration (i.e., 3 mL initial media becomes 300 μL). Next carefully transfer the concentrated supernatant (from the top sample chamber) to a fresh sterile tube. Finally, use the concentrated supernatant for ELISA or cytokine/chemokine detection assays according to manufacturer’s protocol (Figure 2).

Note: If there is low recovery then make sure the centrifugation speed and time are optimized for the molecular weight cutoff of the concentrator filter and choosing a new concentrator with a MWCO at least 2-fold lower than the protein MW may help. For smaller volumes of supernatant, use Pierce Protein Concentrators PES, 0.5 mL capacity.

Problem 4

Higher noise or background signal in ELISA.

Potential solution

Increasing the number of washes during each step of ELISA will reduce the background signal. Washing 3–4 times is essential for better signal to noise ratio.

Problem 5

Too high or too low signal for the standards in ELISA.

Potential solution

Incubate the plates with the proper temperature and required time.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Triparna Sen (triparna.sen@mssm.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by Triparna Sen and Anna Tocheva (triparna.sen@mssm.edu, anna.tocheva@mssm.edu).

Materials availability

This study did not generate new unique reagents. All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

• •This paper does not report original code.
• •The published article includes all datasets analyzed during this study.
• •Any additional information required to analyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by NIH/NCI R01 CA258784 (T.S.), Congressionally Directed Medical Research Programs (DOD-IITRA) LC190161 (T.S.), and Jazz Pharmaceuticals (T.S.).

Author contributions

Conceptualization, T.S.; methodology, S.C., K.V., S.S., R.S., A.T., and T.S.; investigation, S.C., K.V., S.S., R.S., A.T., and T.S.; validation, S.C., K.V., S.S., R.S., A.T., and T.S.; formal analysis, S.C., K.V., S.S., R.S., A.T., and T.S.; resources, T.S.; writing – original draft, S.C., K.V., S.S., R.S., and T.S.; review and editing, all authors; supervision, T.S.; funding acquisition, T.S.

Declaration of interests

T.S. has received research grants from Jazz Pharmaceuticals.