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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Published: June 12th, 2021

DOI:

10.3791/62442

1Institute of Immunology, Third Military Medical University, 2Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Stomatological Hospital, Sun Yat-Sen University, 3Shigatse Branch, Xinqiao Hospital, Third Military Medical University, 4Department of Emergency Medicine, Southwest Hospital, Third Military Medical University, 5Cancer Center, The General Hospital of Western Theater Command

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.

Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

CD8+ T cell-mediated immune response plays a pivotal role in controlling tumor growth. During tumorigenesis, naive CD8+ T cells get activated upon antigen recognition in an MHC class I-restricted manner and subsequently differentiate into effector cells and infiltrate into tumor mass1,2. However, within the tumor microenvironment (TME), prolonged antigen exposure, as well as immunosuppressive factors, drive infiltrated tumor-specific CD8+ T cells into a hyporesponsive state known as "exhaustion"3. Exhausted T cells (Tex) are distinct from effecto....

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All mouse experiments were performed in compliance with the guidelines of the Institutional Animal Care and Use Committees of the Third Military Medical University. Use 6-8-week-old C57BL/6 mice and naïve OT-I transgenic mice weighing 18-22 g. Use both male and female without randomization or "blinding."

1. Preparation of medium and reagents

  1. Prepare cell culture medium D10 as previously described29 by adding 10% fetal bovine serum (FBS), 100 U/m.......

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The schematic of this protocol is shown in Figure 1. Eight days after tumor inoculation, CD45.1+ and CD45.1+CD45.2+ OT-I cells were injected into B16F10-OVA tumor-bearing C57BL/6 mice. The tumor was surgically dissected from CD45.1+ OT-I cell-implanted mice (donor) on day 8 post-transfer and transplanted into tumor-matched CD45.1+CD45.2+ OT-I cell-implanted mice (recipient) in the dorsal flank on the same side as the implanted.......

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T cell-mediated immunity plays a crucial role in immune responses against tumors, with CTLs playing the leading role in eradicating cancerous cells. However, the origins of tumor antigen-specific CTLs within TME have not been elucidated30. The use of this tumor transplantation protocol has provided an important clue that intratumoral antigen-specific CD8+ T cells may not persist for a long time, despite the existence of stem-like TCF1+ progenitor CD8+ T cells. Nota.......

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This study was supported by grants from the National Natural Science Fund for Distinguished Young Scholars (No. 31825011 to LY) and the National Natural Science Foundation of China (No. 31900643 to QH, No. 31900656 to ZW).

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Name Company Catalog Number Comments
0.22 μm filter Millipore SLGPR33RB
1 mL tuberculin syringe KDL BB000925
1.5 mL centrifuge tube KIRGEN KG2211
100 U insulin syringe BD Biosciences 320310
15 mL conical tube BEAVER 43008
2,2,2-Tribromoethanol (Avertin) Sigma T48402-25G
2-Methyl-2-butanol Sigma 240486-100ML
70 μm nylon cell strainer BD Falcon 352350
APC anti-mouse CD45.1 BioLegend 110714 Clone:A20
B16F10-OVA cell line bluefbio BFN607200447
BSA-V (bovine serum albumin) Bioss bs-0292P
BV421 Mouse Anti-Mouse CD45.2 BD Horizon 562895 Clone:104
cell culture dish BEAVER 43701/43702/43703
centrifuge Eppendorf 5810R-A462/5424R
cyclophosphamide Sigma C0768-25G
Dulbecco's Modified Eagle Medium Gibco C11995500BT
EasySep Mouse CD8+ T Cell Isolation Kit Stemcell Technologies 19853
EDTA Sigma EDS-500g
FACS tubes BD Falcon 352052
fetal bovine serum Gibco 10270-106
flow cytometer BD FACSCanto II
hemocytometer PorLab Scientific HM330
isoflurane RWD life science R510-22-16
KHCO3 Sangon Biotech A501195-0500
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation Life Technologies L10199
needle carrier RWD Life Science F31034-14
NH4Cl Sangon Biotech A501569-0500
paraformaldehyde Beyotime P0099-500ml
PE anti-mouse TCR Vα2 BioLegend 127808 Clone:B20.1
Pen Strep Glutamine (100x) Gibco 10378-016
PerCP/Cy5.5 anti-mouse CD8a BioLegend 100734 Clone:53-6.7
RPMI-1640 Sigma R8758-500ML
sodium azide Sigma S2002
surgical forceps RWD Life Science F12005-10
surgical scissors RWD Life Science S12003-09
suture thread RWD Life Science F34004-30
trypsin-EDTA Sigma T4049-100ml

  1. Blank, C. U., et al. Defining 'T cell exhaustion. Nature Reviews Immunology. 19 (11), 665-674 (2019).
  2. Leko, V., Rosenberg, S. A. Identifying and targeting human tumor antigens for T cell-based immunotherapy of solid tumors. Cancer Cell. 38 (4), 454-472 (2020).
  3. McLane, L. M., Abdel-Hakeem, M. S., Wherry, E. J. CD8 T cell exhaustion during chronic viral infection and cancer. Annual Review of Immunology. 37, 457-495 (2019).
  4. Davis, M. M., Brodin, P. Rebooting human immunology. Annual Review of Immunology. 36, 843-864 (2018).
  5. Sharma, P., Allison, J. P. The future of immune checkpoint therapy. Science. 348 (6230), 56-61 (2015).
  6. Littman, D. R. Releasing the brakes on cancer immunotherapy. Cell. 373 (16), 1490-1492 (2015).
  7. Verma, V., et al. PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1(+)CD38(hi) cells and anti-PD-1 resistance. Nature Immunology. 20, 1231-1243 (2019).
  8. Hashimoto, M., et al. CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annual Review of Medicine. 69, 301-318 (2018).
  9. Dammeijer, F., et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell. 38 (5), 685-700 (2020).
  10. Buchwald, Z. S., et al. Tumor-draining lymph node is important for a robust abscopal effect stimulated by radiotherapy. Journal for ImmunoTherapy of Cancer. 8 (2), 000867 (2020).
  11. Philip, M., Schietinger, A. Heterogeneity and fate choice: T cell exhaustion in cancer and chronic infections. Current Opinion in Immunology. 58, 98-103 (2019).
  12. Miller, B. C., et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nature Immunology. 20, 326-336 (2019).
  13. Wu, T. D., et al. Peripheral T cell expansion predicts tumour infiltration and clinical response. Nature. 579, 274-278 (2020).
  14. Im, S. J., Konieczny, B. T., Hudson, W. H., Masopust, D., Ahmed, R. PD-1+ stemlike CD8 T cells are resident in lymphoid tissues during persistent LCMV infection. Proceedings of the National Academy of Sciences of the United State of America. 117 (8), 4292-4299 (2020).
  15. Beltra, J. C., et al. Developmental relationships of four exhausted CD8(+) T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity. 52 (5), 825-841 (2020).
  16. Myers, L. M., et al. A functional subset of CD8(+) T cells during chronic exhaustion is defined by SIRPalpha expression. Nature Communications. 10 (1), 794 (2019).
  17. Jansen, C. S., et al. An intra-tumoral niche maintains and differentiates stem-like CD8 T cells. Nature. 576, 465-470 (2019).
  18. Jadhav, R. R., et al. Epigenetic signature of PD-1+ TCF1+ CD8 T cells that act as resource cells during chronic viral infection and respond to PD-1 blockade. Proceedings of the National Academy of Sciences of the United State of America. 116 (28), 14113-14118 (2019).
  19. Li, H., et al. Dysfunctional CD8 T cells form a proliferative, dynamically regulated compartment within human melanoma. Cell. 176 (4), 775-789 (2018).
  20. Kurtulus, S., et al. Checkpoint blockade immunotherapy induces dynamic changes in PD-1(-)CD8(+) tumor-infiltrating T cells. Immunity. 50 (1), 181-194 (2019).
  21. Fransen, M. F., et al. Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy. JCI Insight. 3 (23), 124507 (2018).
  22. E, J. F., et al. CD8(+)CXCR5(+) T cells in tumor-draining lymph nodes are highly activated and predict better prognosis in colorectal cancer. Human Immunology. 79 (6), 446-452 (2018).
  23. Snell, L. M., et al. CD8(+) T cell priming in established chronic viral infection preferentially directs differentiation of memory-like cells for sustained immunity. Immunity. 49 (4), 678-694 (2018).
  24. Siddiqui, I., et al. Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity. 50 (1), 195-211 (2019).
  25. Wang, Y., et al. The transcription factor TCF1 preserves the effector function of exhausted CD8 T cells during chronic viral infection. Frontiers in Immunology. 10, 169 (2019).
  26. Krishna, S., et al. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science. 370 (6522), 1328-1334 (2020).
  27. Yost, K. E., et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nature Medicine. 25, 1251-1259 (2019).
  28. Zitvogel, L., Pitt, J. M., Daillere, R., Smyth, M. J., Kroemer, G. Mouse models in oncoimmunology. Nature Reviews Cancer. 16 (12), 759-773 (2016).
  29. Li, Y., et al. Bcl6 preserves the suppressive function of regulatory T cells during tumorigenesis. Frontiers in Immunology. 11, 806 (2020).
  30. Yu, D., Ye, L. A portrait of CXCR5(+) follicular cytotoxic CD8(+) T cells. Trends in Immunology. 39 (12), 965-979 (2018).
  31. Bracci, L., et al. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clinical Cancer Research. 13 (2), 644-653 (2007).
  32. Salem, M. L., El-Naggar, S. A., Mahmoud, H. A., Elgharabawy, R. M., Bader, A. M. Cyclophosphamide eradicates murine immunogenic tumor coding for a non-self-antigen and induces antitumor immunity. International Journal of Immunopathology and Pharmacology. 32, 1-5 (2018).
  33. Thorsson, V., et al. The Immune landscape of cancer. Immunity. 48 (4), 812-830 (2018).

Erratum

Erratum: Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

An erratum was issued for: Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice. The Protocol was updated.

Step 6.10 of the Protocol was updated from:

Administer penicillin every 8-12 h after the surgery for 3 days. Monitor the mouse's eating, drinking, moving, and the area operated on. Return the transplant recipient to the company of other animals only after it has fully recovered.
NOTE: The administration of buprenorphine is suggested to prevent post-surgical pain [delete sentence]. The mouse typically recovers from the trauma of the surgery within 3 days. If the mouse is not back to normal feeding and mobility and shows any manifestations of infection, consult a veterinarian for interventions or euthanize it.

to:

Administer buprenorphine subcutaneously at a dose of 0.1 mg/kg body weight every 8 h three times after surgery to alleviate the pain. Monitor the mouse's eating, drinking, moving, and the area operated on. Return the transplant recipient to the company of other animals only after it has fully recovered.
NOTE: The mouse typically recovers from the trauma of the surgery within 3 days. If the mouse is not back to normal feeding and mobility and shows any manifestations of infection, consult a veterinarian for interventions or euthanize it.

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