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).

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 na&#239;ve OT-I transgenic mice weighing 18-22 g. Use both male and female without randomization or &#34;blinding.&#34;
1. Preparation of medium and reagents
<ol>
	<li>Prepare cell culture medium D10 as previously described29 by adding 10% fetal bovine serum (FBS), 100 U/m...

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

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...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62442">Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+&#160;T Cells in Mice</a>. The Protocol&#160;was&#160;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&#39;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&#39;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.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62442">Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+&#160;T Cells in Mice</a>. The Protocol&#160;was&#160;updated.

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

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 &#34;exhaustion&#34;3. Exhausted T cells (Tex) are distinct from effector or memory T cells generated in acute viral infection, both transcriptionally and epigenetically. These Tex cells are mainly characterized by the sustained and elevated expression of a series of inhibitory receptors as well as the hierarchical loss of effector functions. Further, the impaired proliferative capacity of exhausted CD8+ T cells results in decreasing numbers of tumor-specific T cells, such that the residual CD8+ T cells within the TME can barely provide sufficient protective immunity against tumor progression3. Thus, the maintenance or reinforcement of intratumoral antigen-specific CD8+ T cells is indispensable for tumor repression.
Moreover, immune checkpoint blockade (ICB) therapy is believed to reinvigorate Tex in tumors by increasing T cell infiltration and hence, T cell numbers and rejuvenating T cell functions to boost tumor repression. The widespread application of ICB treatment has changed the cancer therapy landscape, with a substantial subset of patients experiencing durable responses4,5,6. Nevertheless, the majority of patients and cancer types do not or only temporarily respond to ICB. Inadequate T cell infiltration in the TME has been postulated to be one of the underlying mechanisms accounting for ICB resistance7,8.
Several studies have demonstrated the heterogeneity of tumor-infiltrating CD8+ T cells (TILs) in both patients and mouse models9,10,11,12. It has been confirmed that a subset of CD8+ T cells expressing T cell factor-1 (TCF1) in a tumor mass exhibits stem cell-like properties, which could further give rise to terminally exhausted T cells and is responsible for the proliferation burst after ICB therapy12,13,14,15,16,17,18,19,20,21,22. However, it has been proved that only a small proportion of antigen-specific TCF1+CD8+ T cells exist in the TME and generate an expanded pool of differentiated progeny in response to ICB23,24,25,26. Whether the limited size of this population is enough to ensure the persistence of cytotoxic T lymphocytes (CTLs) to control tumor progression remains unknown, and whether there is replenishment from periphery tissues requires further investigation. Furthermore, recent research suggests the insufficient reinvigoration capacity of pre-existing tumor-specific T cells and the appearance of novel, previously non-existing clonotypes after anti-programmed cell death protein 1 treatment. This indicates that T cell response to checkpoint blockade may be due to the new influx of a distinct repertoire of T cell clones27. Together with the presence of bystander non-tumor-reactive cytotoxic T cell fraction in the TME, these findings prompted the establishment of a tumor allograft model to study the role of periphery-derived CD8+ T cells11.
Until now, several kinds of tumor implantation, as well as immune cell adoptive transfer, have been widely used in the field of tumor immunology28. TILs, peripheral blood mononuclear cells, and tumor-reactive immune cells originated from other tissues can be well-characterized using these methods. However, when studying the interactions between systemic and local antitumor immunity, these models appear inadequate to examine the interactions between immune cells derived from the periphery and the TME. Here, tumor tissues were transplanted from donors into tumor-matched recipient mice to precisely trace the influx of recipient-derived immune cells and observe the donor-derived cells in the TME concomitantly.
In this study, a murine syngeneic model of melanoma was established with the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen ovalbumin. 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 MHC molecule H2-Kb, enable the study of antigen-specific T cell responses developed in the B16F10-OVA tumor model. Combining this model with tumor transplantation, the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells were compared to reveal a dynamic transition between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in the TME, which sheds new light on the dynamics of tumor-specific T cell immune responses in the TME.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tuberculin syringe</td>
			<td>KDL</td>
			<td>BB000925</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>KIRGEN</td>
			<td>KG2211</td>
			<td></td>
		</tr>
		<tr>
			<td>100 U insulin syringe</td>
			<td>BD Biosciences</td>
			<td>320310</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>BEAVER</td>
			<td>43008</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2,2-Tribromoethanol (Avertin)</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Methyl-2-butanol</td>
			<td>Sigma</td>
			<td>240486-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon cell strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45.1</td>
			<td>BioLegend</td>
			<td>110714</td>
			<td>Clone:A20</td>
		</tr>
		<tr>
			<td>B16F10-OVA cell line</td>
			<td>bluefbio</td>
			<td>BFN607200447</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-V (bovine serum albumin)</td>
			<td>Bioss</td>
			<td>bs-0292P</td>
			<td></td>
		</tr>
		<tr>
			<td>BV421 Mouse Anti-Mouse CD45.2</td>
			<td>BD Horizon</td>
			<td>562895</td>
			<td>Clone:104</td>
		</tr>
		<tr>
			<td>cell culture dish</td>
			<td>BEAVER</td>
			<td>43701/43702/43703</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R-A462/5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>cyclophosphamide</td>
			<td>Sigma</td>
			<td>C0768-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse CD8+ T Cell Isolation Kit</td>
			<td>Stemcell Technologies</td>
			<td>19853</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>flow cytometer</td>
			<td>BD</td>
			<td>FACSCanto II</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>PorLab Scientific</td>
			<td>HM330</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>RWD life science</td>
			<td>R510-22-16</td>
			<td></td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td>Sangon Biotech</td>
			<td>A501195-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation</td>
			<td>Life Technologies</td>
			<td>L10199</td>
			<td></td>
		</tr>
		<tr>
			<td>needle carrier</td>
			<td>RWD Life Science</td>
			<td>F31034-14</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sangon Biotech</td>
			<td>A501569-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0099-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse TCR V&#945;2</td>
			<td>BioLegend</td>
			<td>127808</td>
			<td>Clone:B20.1</td>
		</tr>
		<tr>
			<td>Pen Strep Glutamine (100x)</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5 anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100734</td>
			<td>Clone:53-6.7</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma</td>
			<td>R8758-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>RWD Life Science</td>
			<td>F12005-10</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>RWD Life Science</td>
			<td>S12003-09</td>
			<td></td>
		</tr>
		<tr>
			<td>suture thread</td>
			<td>RWD Life Science</td>
			<td>F34004-30</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049-100ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

tumor transplantation for assessing the dynamics of tumor-infiltrating cd8+ t cells in mice

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.

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...

Watch this Scientific Journal Video about Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice at JoVE.com

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

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.

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

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

This method can help answer the key questions in tumor immunology, including whether there is a replenishment of tumor-specific immune cells from peripheral tissues, the transitions between newly infiltrated and already existing immune cells within tumor microenvironment, and their responses to immunotherapy. This is convenient and easy-to-follow technique to distinguish the periphery-derived cells from tumor-infiltrated immune cells and track the dynamic, phenotypic, and functional changes of these cells in longitudinal assays. Begin by withdrawing 100 microliters of the prepared B16F10-OVA cell suspension into a 1 mL tuberculin syringe.Tap the barrel to move any bubbles to the top and gently push the plunger to remove air bubbles. Restrain the mouse to expose its abdomen. Press the left hind leg with the little finger to tighten the skin of the left inguinal region.Remove the mouse's hair from its left lower abdomen with an electric shaver. Use cotton soaked in 75%ethanol to clean the posterior quadrant of the left abdomen. Hold the syringe at a shallow angle between 0 to 15 degrees and, with the bevel of the needle facing upwards, insert it at the side of the left upper thigh.Advance the needle 0.5 to 1 centimeter through the subcutaneous tissue into the inguinal region. Pull back on the plunger before injecting. If there is negative pressure, depress the plunger entirely and observe the formation of a small fluid pocket, or a bolus, in the subcutis.Remove the needle after the injection is carried out. Release and place the mouse back into the cage. On day six to eight after B16F10-OVA implantation, measure the tumor size with a vernier scale.Select the mice with an approximately three-millimeter diameter or a mung bean-sized tumor, and divide them equally and randomly into two groups. Withdraw 200 microliters of OT-1 cell suspension into a 100-unit 29-gauge insulin syringe and remove bubbles. Place the mouse separately in a cage, with an infrared lamp over the cage for 5 to 10 minutes to dilate the tail vein.Immobilize the mouse with a restraining device of appropriate size. Hold the tail to straighten it and spray 75%ethanol to make the vein visible. Hold the syringe parallel to the vein and insert it into the vein at an angle of 0 to 15 degrees.Pull back the plunger slightly, and if blood enters the barrel, slowly and steadily inject the suspension at a rate of no more than one milliliter per minute. After the injection is completed, remove the syringe and press the injection area gently for three to five seconds to stop the bleeding. Return the mouse to the cage and closely observe it for a few minutes for adverse reactions.If it has normal mobility and nasal discharge, place it back in the company of the other mice. 8 to 10 days after the adoptive transfer, select donor mice bearing comparable tumor mass of approximately five-millimeter diameter for transplantation surgery. Place a 100-millimeter-by-20-milliliter dish in a biosafety cabinet and add 10 milliliters of sterile, ice-cold PBS.After euthanizing the mouse, immerse it in 75%ethanol for three to five minutes. Place the mouse in the supine position on a dissection board covered with clean absorbent paper in the biosafety cabinet. Restrain the mouse limbs with dissection needles.Cut the skin along the midline from above the urethral orifice to the xiphoid with scissors. Stretch the skin towards the left side of the mouse body with tweezers and restrain the skin with dissection needles. Excise the tumor, keeping its capsule as intact as possible.Carefully and gently remove the connective tissue near the tumor with surgical scissors, then place the tumor tissue in the dish containing sterile, ice-cold PBS for subsequent transplantation. Use veterinary ointment on eyes to prevent them from drying. Shave the left flank of the mouse with an electric shaver, then apply a depilatory cream to remove the remaining hair.Place the mouse inside the biosafety cabinet in a prone position on a dissection board covered with clean absorbent paper, with the mouse's vertical axis parallel to and its head to the right side of the experimenter. Rub the skin of the shaved area with cotton soaked in povidone-iodine. Lift the skin at the midpoint between the mouse hip joints with surgical tweezers.Use the scissors to make a five-millimeter-long vertical excision and extend the cut rostrally along the dorsal midline to around 10 to 15 millimeters. Perform a sharp dissection by inserting the closed tips of the scissors into the incision and then opening to separate the peritoneum of the left flank from the skin and soft tissue. Perform sharp dissection several times to generate a skin pocket on the left flank.Deposit the encapsulated, intact, donor-derived tumor mass into the pocket. Close the incision by an interrupted suture, using two or three stitches for each incision. Disinfect the skin around the cut with cotton soaked in povidone-iodine.Place the mouse in the lateral position in a clean and warm cage. Monitor it continuously until it has regained sufficient consciousness to maintain sternal recumbency. Administer buprenorphine subcutaneously at a dose of 0.1 milligram per kilogram of body weight every eight hours, for a total of three doses.Return the transplant recipient to the company of other animals only after it has fully recovered. Using flow cytometry, two populations of CD44-negative and CD8-positive tumor antigen-specific T cells were easily identified in the tumor microenvironment, including CD45.1-positive donor-derived and CD45.1-negative and CD45.2-positive recipient-derived tumor-infiltrating CD8-positive T cells. At day two post-transplantation, there were approximately 83%of donor-derived antigen-specific CD8-positive T cells within the transplanted tumor, more predominant than their recipient-derived counterparts.The proportion of recipient-derived OT-1 cells was elevated in the late stage of tumorigenesis, exceeding tumor-inherent OT-1 cells derived from the donor. The most important thing to remember is that to perform a gentle dissection during subcutaneous tumor transplantation to avoid injuries on surrounding tissues, especially in granular lymph node.

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JoVE Journal

Cancer Research

6.0K Views.  Third Military Medical University. This method can help answer the key questions in tumor immunology, including whether there is a replenishment of tumor-specific immune cells from peripheral tissues, the transitions between newly infiltrated and already existing immune cells within tumor microenvironment, and their responses to immunotherapy. This is convenient and easy-to-follow technique to distinguish the periphery-derived cells from tumor-infiltrated immune cells and track the dynamic, phenotypic, and functional changes o...