Protocol for investigating the impact of transcription regulator deficiency on tumor-specific CD8⁺ T cell responses via adoptive cell transfer

Summary

Transcription factors play a crucial role in the differentiation of tumor-specific CD8⁺ T cells and significantly influence their capacity to repress tumors. Here, we present a protocol for creating a transcription regulator inhibitor of DNA 3 (ID3) conditional knockout mouse in tumor-specific CD8⁺ T cells, induced by tamoxifen. We describe steps for examining the impact of ID3 deficiency on the differentiation of tumor-specific memory CD8⁺ T cells (Ttsm) and progenitors of exhausted CD8⁺ T cells (Tpex) in tumor-draining lymph nodes through a co-adoptive transfer assay.

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

Graphical abstract

Highlights

• •Instructions for the establishment of the B16.GP melanoma mouse model
• •Steps for preparing Id3^−/− P14 and Id3^fl/fl P14 cells by the administration of tamoxifen
• •Guidance on investigating the effect of Id3 deficiency on CD8⁺ T cell differentiation

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

Transcription factors play a crucial role in the differentiation of tumor-specific CD8⁺ T cells and significantly influence their capacity to repress tumors. Here, we present a protocol for creating a transcription regulator inhibitor of DNA 3 (ID3) conditional knockout mouse in tumor-specific CD8⁺ T cells, induced by tamoxifen. We describe steps for examining the impact of ID3 deficiency on the differentiation of tumor-specific memory CD8⁺ T cells (Ttsm) and progenitors of exhausted CD8⁺ T cells (Tpex) in tumor-draining lymph nodes through a co-adoptive transfer assay.

Before you begin

Both Ttsm and Tpex cells are key responders to immune checkpoint blockade (ICB) therapy, and their generation can significantly enhance the efficacy of this treatment.^2,3,4,5,6 The protocol described here investigates the effect of transcription regulator inhibitor of DNA 3 (ID3) on the differentiation of both Ttsm and Tpex cells in tumor-draining lymph nodes (TdLNs) and the tumor microenvironment (TME). Additionally, this protocol can be adapted to explore the roles of other transcription factors in the differentiation and maintenance of various tumor-specific CD8⁺ T cell subsets.

Institutional permissions

All mouse experiments and feeding conditions were conducted in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of the Chongqing Medical University. If your experiments involve animals, you must obtain the necessary institutional permissions.

Construction of ERT2^Cre-Id3^fl/fl mice

Timing:Over 24 weeks

• 1.Cross ERT2^Cre mice, in which Cre recombinase expression is induced by tamoxifen, with Id3^fl/fl mice, which contain loxP sequences inserted before exon 1 and after exon 2.
• 2.
  Genotype ERT2^Cre-Id3^fl/fl mouse using PCR.

  • a.
    Obtain a small piece of tissue (approximately 1–2 mm) from the toes of the mouse (3–4 weeks age) and place it in a PCR tube.
  • b.
    Add 100 μL of 0.05 M NaOH to the PCR tube and incubate the tissue at 98°C for 1.5 h in a water incubator to dissolve the tissue.
  • c.
    After cooling to room temperature, add 20 μL of 1 M Tris HCl (pH 7.4) buffer to the PCR tube.
  • d.
    Centrifuge the PCR tube briefly to collect the supernatant, which will serve as the DNA template.
  • e.
    Add 2 μL of the supernatant to a PCR mixture containing primers specific for ERT2^Cre and Id3, and amplify the product using a thermal cycler.

    Note: The forward primer for ERT^cre is 5′-CCACGACCAAGTGACAGCAATG-3′, and the reverse primer is 5′-CAGAGACGGAAATCCATCGCTC-3'. The forward primer for Id3^fl/fl is 5′-CCACGACCAAGTGACAGCAATG-3′, and the reverse primer is 5′-CCATTTGGTTCTATG TATGCCCGTG-3'.

  • f.
    Analyze the PCR products using gel electrophoresis at an appropriate concentration.

Construction of P14-ERT2^Cre-Id3^fl/fl mice (CD45.1/ CD45.2)

Timing:Over 24 weeks

• 3.Cross ERT2^Cre-Id3^fl/fl mice on a CD45.2 background with P14 mice on a CD45.1 background (Figure 1).

Figure 1.

The process diagram for the establishment of P14-ERT2^Cre-Id3^fl/fl mice (CD45.1/ CD45.2)

Genotype the resulting P14-ERT2^Cre-Id3^fl/fl mice (CD45.1/ CD45.2) using PCR as described above.

• 4.
  Phenotype P14-ERT2^Cre-Id3^fl/fl mice (CD45.1/ CD45.2) using FACS.

  • a.
    Cut the tail of the mouse and collect 3–4 drops of blood into 1 mL of ACK (Ammonium-Chloride-Potassium) lysis buffer).
  • b.
    Centrifuge the samples at 800 × g for 3 min, discard the supernatant, and resuspend the cells in 200 μL of FACS solution (PBS containing 2% FBS and 0.1 mg/mL NaN3).
  • c.
    Prepare the antibody mixture in FACS solution.

    Note: P14 cells are characterized as Vα2⁺CD8⁺, so the antibody mixture should include Live/Dead stain, anti-Vα2, anti-CD44, anti-CD8, anti-CD45.1, and anti-CD45.2 antibodies.

  • d.
    Incubation of sample with antibody mixture.

    • i.
      Add 200 μL of the sample to each well of a 96-well plate and centrifuge the samples at 800 × g for 1 min.
    • ii.
      Discard the supernatant and resuspend the cells in 50 μL of the antibody mixture in each well.
    • iii.
      Incubate on ice for 30 min in the dark.
  • e.
    After incubation, wash the cells twice with FACS solution and then resuspend them in 200 μL of FACS solution.
  • f.
    Analyze the P14 cells (Live/Dead^-Vα2⁺CD8⁺) among total lymphocytes using a BD Fortessa cell flow cytometer.

    • i.
      Gate the Live/Dead negative staining cells as the Live/Dead^- cells.
    • ii.
      Gate Vα2⁺CD8⁺ CD44^- cells in Live/Dead^- cells as P14 cells.

      Note: If the percentage of Vα2⁺CD8⁺ cells in total live lymphocytes exceeds 85%, the mouse is identified as P14-ERT2^Cre-Id3^fl/fl mice (CD45.1/ CD45.2).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
PerCP/Cyanine5.5 anti-mouse CD8a (clone 53–6.7) (diluted by 1:300)	BioLegend	Cat# 100734; RRID:AB_2075238
PerCP/Cyanine5.5 anti-mouse CD44 (clone IM7) (diluted by 1:300)	BioLegend	Cat# 103032; RRID:AB_2076204
PE/Cyanine7 anti-mouse CD45.1 (clone A20) (diluted by 1:400)	BioLegend	Cat# 110730
Alexa Fluor 700 anti-mouse CD45.2 (clone 104) (diluted by 1:300)	BioLegend	Cat# 109822; RRID: AB_493731
PE mouse Anti-ID3 (clone S30-778) (diluted by 1:400)	BD Biosciences	Cat# 556537; RRID:AB_396457
TCF1/TCF7 (C63D9) rabbit mAb (Alexa Fluor 488 Conjugate)#6444 (clone C63D9) (diluted by 1:400)	Cell Signaling Technology	Cat# 2203; RRID: N/A
PE TOX monoclonal antibody (clone TXRX10) (diluted by 1:200)	Invitrogen	Cat# 12-6502-82; RRID: AB_10855034
PE anti-mouse TCR Vα2 (clone B20.1) (diluted by 1:300)	BioLegend	127808
Brilliant Violet 510 anti-mouse CD8a (clone 53–6.7) (diluted by 1:300)	BioLegend	Cat# 100752; RRID:AB_2563057
APC anti-mouse CD45.1 (clone A20) (diluted by 1:300)	BioLegend	Cat# 110714; RRID: AB_313503
TruStain FcX PLUS (anti-mouse CD16/32) antibody (clone S17011E) (diluted by 1:200)	BioLegend	Cat# 156603; RRID: AB_2783137
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM)	Gibco	Cat# 11965092
RPMI 1640	Gibco	Cat# 11875093
Fetal bovine serum (FBS), Premium Plus	Gibco	Cat# A5669701
Trypsin-EDTA (0.25%), phenol red	Gibco	Cat# 25200056
Non-essential amino acids (MEM NEAA) (100×)	Gibco	Cat# 11140050
Sodium pyruvate (100 mM)	Gibco	Cat# 11360070
Tamoxifen	Sigma	Cat# T5648
Penicillin-Streptomycin-Glutamine solution	Gibco	Cat# 10378016
Percoll	Cytiva	Cat# 17089101
Cyclophosphamide (CTX)	Sigma	Cat# PHR1404
Critical commercial assays
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit	Thermo Fisher Scientific	Cat# L34975
Foxp3/Transcription Factor Staining Buffer Kit	eBioscience	00–5523
Experimental models: Cell lines
B16.Gp cells	Beijing Biocytogen Co., Ltd., China	Custom
Experimental models: Organisms/strains
Mouse: C57BL/6J (CD45.2) (male, age of 6–8 weeks)	The Jackson Laboratory	Stock No: 000664
Mouse: B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) (male, age of 6–8 weeks)	Jackson Laboratory	Stock No: 002014
Mouse: B6.129S6(Cg)-Id3tm2.1Zhu/J (Id3fl/fl) (male, age of 6–8 weeks)	Jackson Laboratory	Stock No: 024496
Mouse: ERT2Cre (male, age of 6–8 weeks)	Presented by Prof. Yisong Wan	N/A
Software and algorithms
Prism GraphPad	GraphPad Software	Version 10.2.3 https://www.graphpad.com
FlowJo	Tree Star	https://www.flowjo.com/; RRID: SCR_008520
Others
70 μm Nylon mesh	BD Falcon	352350

Materials and equipment

Recipe for reagents

ACK lysis buffer

Reagent	Final concentration	Amount
KH4Cl	8.3 mg/mL	1.66 g
NaHCO3	1.0 mg/mL	200 mg
EDTA	0.029 mg/mL	5.8 mg
ddH2O	N/A	supplement to 200 mL
Total	N/A	200 mL

Store at 4°C for up to 12 months.

D10 medium

Reagent	Final concentration	Amount
FBS	10%	100 g
Penicillin/streptomycin	1%	10 g
Non-essential amino acids	1%	10 g
Pyruvic acid	1%	10 g
DMEM	N/A	supplement to 1 L
Total	N/A	1,000 mL

Store at 4°C for up to 1 month.

Step-by-step method details

In vitro culture of melanoma cell line B16.GP

Timing: 2–3 days

This step outlines the in vitro culture of the melanoma cell line B16.GP.

• 1.
  Thaw B16.GP(B16F10.GP-Luciferase) cells.

  • a.
    Place a frozen vial containing B16.GP cells in a 37°C water bath and gently agitate the tube to thaw the frozen cells.

    Note: B16.GP cells are derived from the B16F10 melanoma cell line, with the LCMV Cl13 glycoprotein gene sequence integrated into the B16F10 genome via CRISPR-Cas9 technology.

  • b.
    Transfer the thawed cells into a 15 mL tube, wash with 3–4 mL of D10 medium, and centrifuge at 300 × g for 5 min.
  • c.
    Discard the supernatant and resuspend the cells in D10 medium.
• 2.Culture B16.GP Cells: Culture the B16.GP cells in complete DMEM-10 medium supplemented with 100 U/mL puromycin in an incubator set to 37°C with 5% CO₂. Change the culture medium every 2–3 days.

CRITICAL: The addition of 100 U/mL puromycin is necessary to select for B16.GP cells that express the LCMV Cl13 glycoprotein.

• 3.Passaging Cells: When the cells reach 80–90% confluency, detach them using 0.25% trypsin-EDTA. After detaching the cells, add fresh culture medium to neutralize the trypsin and passage the cells into new culture vessels at a 1:3 ratio.

Establish the B16.GP melanoma mouse model

Timing: 7–10 days

This step outlines the procedure for constructing a subcutaneous B16.GP melanoma mouse model.

• 4.Detach and Prepare B16.GP Cells: Detach the in vitro cultured B16.GP cells with 0.25% trypsin-EDTA, then wash and count the cells.
• 5.Inject B16.GP Cells: Subcutaneously inject 1 × 10⁶ of B16.GP cells in 100 μL of DMEM-10 into the right groin of each mouse using a 25- to 30- gauge needle.
• 6.Monitor Tumor Growth: Measure the length of both the long and short axes of the tumor every 2–3 days to monitor growth. Calculate the tumor volume using the formula: (length x width²) / 2.

Note: As B16.GP cells express luciferase, the growth of B16.GP cells on mice can also be evaluated by in vivo bioluminescence imaging system, such as the IVIS Spectrum or similar imaging systems.

Prepare Id3^−/− P14 and Id3^fl/fl P14 cells

Timing: 6 days

This step describes the preparation of Id3^−/− P14 cells and Id3^fl/fl P14 cells (Figures 2 and 3).

• 7.Tamoxifen Injection: Intraperitoneally inject tamoxifen at a dosage of 1, 2, or 4 mg/day/mouse into P14-ERT2^Cre-Id3^fl/fl mice (CD45.1⁺ CD45.2⁺) or P14-Id3^fl/fl mice (CD45.1⁺) for four consecutive days.
• 8.
  Isolation of Lymphocytes:

  • a.
    Euthanasia the P14-ERT2^Cre-Id3^fl/fl mice or P14-Id3^fl/fl mice by carbon dioxide (CO₂) inhalation one day after tamoxifen treatment.
  • b.
    Manually isolate spleens and lymph nodes from the P14-ERT2^Cre-Id3^fl/fl mice (CD45.1⁺ CD45.2⁺) and P14-Id3^fl/fl mice (CD45.1⁺), respectively.
  • c.
    Grind the spleens on a sterilized 70 μm nylon mesh using 2 mL of ACK lysis buffer.

    • i.
      After completely grinding the spleens, add 3–5 mL of R2 (RPMI 1640 medium with 2% FBS) medium.
    • ii.
      Add the isolated lymph nodes to the same filter and continue grinding in the R2 solution.
  • d.
    Centrifuge the samples at 800 × g for 3 min, discard the supernatant, and resuspend with cells in FACS solution on ice.
• 9.
  Analyze Id3 Expression in P14 Cells:

  • a.
    Block Fc receptors of lymphocytes.

    • i.
      Centrifuge the samples at 800 × g for 3 min, discard the supernatant, and resuspend cells with 100 μL anti-CD16/32 in Eppendorf tube.
    • ii.
      Incubate on ice for 30 min in the dark.
    • iii.
      After incubation, cells were centrifugated 800 × g for 3 min, and discard the supernatant.
  • b.
    Stain the single-cell suspension with an antibody mixture containing Live/Dead stain, anti-Vα2, anti-CD44, anti-CD8, anti-CD45.1, and anti-CD45.2, as described in step 4c.
  • c.
    Wash the cells twice using FACS solution.
  • d.
    Stain the cells with anti-Id3 using the Foxp3/Transcription Factor Staining Buffer Kit.

    • i.
      Add 150 μL of Fixation/Permeabilization solution to each well according to the reagent instructions, mixing the sample gently.
    • ii.
      Incubate at room temperature for 30 min in the dark, then wash twice with 1× Fixation/Permeabilization wash buffer.
    • iii.
      Prepare the intracellular marker antibody mixture specific for the corresponding nuclear molecules using 1× Fixation/Permeabilization wash buffer.
    • iv.
      Add 50 μL of this antibody mixture to each well and incubate at room temperature for 30 min in the dark.
    • v.
      After incubation, wash the lymphocyte samples twice with 1× Fixation/Permeabilization wash buffer.
  • e.
    Resuspend the cells in 200 μL/well of FACS solution, and analyze the expression of Id3 in ERT2^Cre-Id3^fl/fl and Id3^fl/fl P14 cells using a BD fortessa cell flow cytometer.

    Note: It is crucial to identify the optimal tamoxifen dose for these experiments. The selected dose should balance maximal efficiency in inducing conditional Id3 knockout in P14 cells with minimal toxicity to the mice. In our experiment, 2 mg was found to be the most appropriate dose of tamoxifen.

Figure 2.

The process diagram for preparing Id3^−/− P14 and Id3^fl/fl P14 cells

Figure 3.

Representative FACS analysis of ID3 expression in P14 cells from P14-ERT2^Cre-Id3^fl/fl mice and P14-Id3^fl/fl mice following tamoxifen treatment

Co-adoptive transfer of Id3^−/− P14 and Id3^fl/fl P14 cells

Timing: 1.5 days

This step outlines the procedure for the co-adoptive transfer of Id3^−/− P14 and Id3^fl/fl P14 cells into B16.GP tumor-bearing mice.

• 10.Cyclophosphamide Injection: On day −1, intraperitoneally inject tumor-bearing mice with 4 mg/mouse of cyclophosphamide (CTX) the night before the tumor cells reach a diameter of approximately 3–5 mm, which typically occurs about 7–10 days after tumor cell inoculation.

Note: The administration of CTX is intended to deplete endogenous lymphocytes, thereby promoting the homing of transferred lymphocytes to secondary lymphoid organs and the tumor mass.^7,8

• 11.Cell Preparation: Calculate the total number of Id3^−/− P14 and Id3^fl/fl P14 cells. Use an equal number of Id3^−/− P14 or Id3^fl/fl P14 cells, and suspend these cells in 500 μL of RPMI 1640 medium.
• 12.Intravenous Injection: Restrain the B16.GP tumor-bearing mice using Flat-lying mouse squeeze holder, then inject 5 × 10⁵ of either Id3^−/− P14 or Id3^fl/fl P14 cells in 500 μL of RPMI 1640 medium intravenously into the tail vein of mice using insulin syringe (0.33∗13 mm(29G)U-100).

CRITICAL: It is essential to perform the co-adoptive transfer using equal numbers of Id3^−/− P14 or Id3^fl/fl P14 to effectively compare their differentiation capacities.

CRITICAL: In this co-adoptive transfer assay, it is essential to use P14 cells with different genetic backgrounds. The P14-ERT2^Cre-Id3^fl/fl mice have a CD45.1⁺ CD45.2⁺ background, while the P14-Id3^fl/fl mice have a CD45.1⁺ background. This distinction allows for the differentiation of P14 cells from P14-ERT2^Cre-Id3^fl/fl mice and P14-Id3^fl/fl mice by detecting CD45.1⁺ or CD45.2⁺ after adoptive transfer into the same mouse.

Determine the effect of Id3 on the differentiation of Ttsm and Tpex by FACS analysis

Timing: 5–6 h

This step outlines the procedure for analyzing Ttsm and Tpex in TdLNs and TME using FACS (Figures 4 and 5).

• 13.On day 4 or 7, Euthanize the tumor-bearing mice.
• 14.
  Preparation of Single-Cell Suspension from TdLNs.

  • a.
    Manually isolate the TdLNs from the side of the tumor mass.
  • b.
    Grind the lymph nodes using two slides in R2 solution to obtain a single-cell suspension of lymphocytes.
• 15.
  Preparation of Single-Cell Suspension from Tumor-Infiltrating Lymphocytes (TIL) in the TME.

  • a.
    Manually scrape the B16.GP tumor to collect cells.
  • b.
    Pass the cells through a 70 μm nylon filter to create a single-cell suspension.
  • c.
    Purify the TILs by gradient centrifugation using 67% high-concentration Percoll and 44% low-concentration Percoll solutions for 1260 × g for 30 min at room temperature without a break.
  • d.
    After centrifugation, pipet the upper layer of floating tissues, and carefully take the layer of lymphocytes in the middle layer using a sterile pipette.
• 16.
  FACS Analysis of Tumor-Specific CD8⁺ T Cell Subsets in TdLNs and TME.

  • a.
    Transfer single-cell samples from TdLNs and TME into a 96-well circular bottom plate, centrifuge at 800 × g for 1 min, and wash twice with FACS solution.
  • b.
    The Fc receptors of cells were blocked as described in step 9a.
  • c.
    Prepare a surface marker antibody mixture in FACS solution.

    Note: Ttsm cells are characterized as TCF-1⁺ TOX^-, while Tpex cells are TCF-1^- TOX⁺. Additionally, Id3^−/− P14 cells are CD45.1⁺ CD45.2⁺ background, whereas Id3^fl/fl P14 cells are CD45.1⁺ background. The antibody mixture should include anti-CD45.1, anti-CD45.2, anti-CD44, and anti-CD8 to distinguish the differentiation capabilities of Id3^−/− and Id3^fl/fl P14 cells into Ttsm and Tpex.

  • d.
    Add 50 μL of the antibody mixture to each well of the 96-well plate and incubate on ice for 30 min in the dark.
  • e.
    FACS analysis of intracellular markers using Foxp3/Transcription Factor Staining Buffer Kit as described in step 9d, except for the different antibody mixture was used.

    Note: The antibody mixture should include anti-TCF-1 and anti-TOX to distinguish Ttsm (TCF-1⁺TOX^-) and Tpex (TCF-1⁻TOX⁺) cells.

  • f.
    Analyze the subsets of tumor-specific CD8+ T cells using a BD Fortessa flow cytometer.

    • i.
      Gate the Live/Dead negative staining cells as the Live/Dead^- cells.
    • ii.
      Gate CD45.1⁺CD44⁺ cells in Live/Dead⁻ cells.
    • iii.
      Gate CD45.1⁺ CD45.2⁻ and CD45.1⁺ CD45.2⁺ cells as Id3^fl/fl P14 and Id3^−/− P14 cells, respectively.
    • iv.
      In both Id3^fl/fl P14 and Id3^−/− P14 cells, gate TCF-1⁺TOX⁻ and TCF-1⁻TOX⁺ cells as Ttsm and Tpex.

Figure 4.

The process diagram for FACS analysis of Ttsm and Tpex cells in TdLNs following the co-adoptive transfer of Id3^−/− P14 and Id3^fl/fl P14 cells

Figure 5.

Representative FACS analysis of Ttsm and Tpex cells in TdLNs following the co-adoptive transfer of Id3^−/− P14 and Id3^fl/fl P14 cells

Expected outcomes

After administering a 2 mg tamoxifen injection per mouse for four consecutive days to P14-ERT2^Cre-Id3^fl/fl mice and P14-Id3^fl/fl mice, the expression of Id3 in P14 cells isolated from P14-ERT2^Cre-Id3^fl/fl mice is completely abolished, while it remains intact in P14-Id3^fl/fl mice (Figure 4). Subsequently, an equal number of Id3^−/− P14 cells (5×10⁵) or Id3^fl/fl P14 cells (5×10⁵) are co-adoptively transferred into B16.GP melanoma-bearing mice. Both Ttsm and Tpex cells differentiated from Id3^−/− P14 cells and Id3^fl/fl P14 cells are analyzed by FACS. Notably, Ttsm and Tpex cells differentiated from Id3^−/−P14 cells are comparable to those from Id3^fl/fl P14 cells at the early stage of tumor development.

Limitations

This protocol is designed to investigate the effect of transcription factors on the differentiation of tumor-specific CD8⁺ T cells using the B16.GP melanoma cell line and P14 transgenic mice. Researchers may also utilize OT-1 transgenic mice, in which case the B16.GP cell line should be replaced with B16.OVA.

Troubleshooting

Problem 1

Is it necessary to generate homozygous P14-ERT2^Cre-Id3^fl/fl mice with a CD45.2 background?

Potential solution

While it is preferable to generate homozygous P14-ERT2^Cre-Id3^fl/fl mice in a CD45.2 background, this process may be time-consuming. Constructing heterozygous P14-ERT2^Cre-Id3^fl/fl mice in a CD45.2/CD45.1 background is sufficient to distinguish between Id3^−/− P14 and Id3^fl/fl P14 cells after adoptive transfer into the same mouse.

Problem 2

The sizes of melanoma can vary significantly between mice, which can greatly impact the results.

Potential solution

It is advisable to have a single technician establish the subcutaneous melanoma mouse model to minimize mechanical errors and enhance consistency. If there is still significant variability in tumor size, select mice with similar tumor sizes and regroup them accordingly.

Problem 3

P14-ERT2^Cre-Id3^fl/fl mice and P14-Id3^fl/fl mice exhibit mortality following tamoxifen treatment.

Potential solution

It is recommended to determine the optimal concentration of tamoxifen that can effectively induce the conditional knockout of Id3 in P14 cells while minimizing any significant toxic effects on the mice.

Problem 4

The purity of Id3^−/− P14 and Id3^fl/fl P14 cells is not sufficiently enough.

Potential solution

To improve the purity of Id3^−/− P14 and Id3^fl/fl P14 cells, it is advisable to enrich P14 cells using MACS MicroBeads or to sort them by FACS.

Problem 5

Can Ttsm cells be detected within the TME?

Potential solution

Ttsm cells are predominantly found in TdLNs; however, we also identified TCF-1^-TOX⁺CD8⁺ T cells in the TME, which are often referred to as Ttsm-like cells.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Qizhao Huang (huangqizhaotcell@cqmu.edu.cn).

Technical contact

Requests for additional information about the protocol should be directed to Dr. Luming Xu (Xu_LMing@163.com).

Materials availability

This study did not generate new, unique reagents.

Data and code availability

There are no additional data associated with this protocol.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation for Outstanding Young Scholars of China (no. 82322031 to Q.H.), Natural Science Foundation for Outstanding Young Scholars of Chongqing (no. CSTB2024NSCQ-JQX0008 to Q.H.), National Key Research and Development Program of China (no. 2021YFC2300602 to L.Y.), Natural Science Foundation of Chong Qing (no. CSTB2023NSCQ-LZX0010 to Q.H.), and Natural Science Foundation of Guangdong (no. 2024A1515010375 to Q.H.).

Author contributions

Conceptualization, Q.H.; methodology, L.X., Y.W., M.R., and W.L.; writing – original draft, L.X.; writing – review and editing, Q.H.; funding acquisition and supervision, Q.H. and L.Y.

Declaration of interests

The authors declare no competing interests.