We thank Alan B. Hoofring and Erina H. He for graphical assistance. This research was supported by the Intramural Research Program of the National Cancer Institute (ZIA BC010763) and the Intramural NCI Cancer Moonshot Initiative for Cell-based Cancer Immunotherapy.

1. Culturing Human iPSCs (hiPSCs) on Mouse Embryonic Fibroblasts (MEF)
NOTE: Alternative methods for culturing hiPSCs can also be used, including but not limited to: seeding onto a 6 well plate pre-coated with gelatin, a gelatinous protein mixture, recombinant laminin 511, or any other extracellular matrix used in hiPSC expansion, and cultured using defined media specially formulated for human pluripotent stem cell culture.
<ol>
	<li>Culturing MEF
	<ol>
		<li>Coat a 10 cm cell culture Petri dish with 4 mL of 0.1% gelatin and incubate for 30 min at 37 &#176;C.</li>
		<li>Thaw a vial of 4 x 106 irradiated MEF quickly into 10 mL of 37 &#176;C MEF media (DMEM + 10% FBS + 1x penicillin-streptomycin + 1x L-glutamine supplement). Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend the cell pellet in 9 mL of MEF media.</li>
		<li>Remove the gelatin-coated dish from the incubator. Aspirate gelatin and add 7 mL of MEF media. Plate 3 mL of MEF suspension (from step 1.1.2) onto the gelatin-coated dish. Rock the dish side-to-side and front-to-back to ensure even distribution of MEF over the dish. Incubate at 37 &#176;C for 8-36 h.</li>
	</ol>
	</li>
	<li>Passaging hiPSC on MEF 
	NOTE: The data was generated using MART-1 iPSC derived from long-term cultured melanoma TIL, which specifically recognize MART-1 peptide in the context of HLA-A*02:01, as previously described18.&#160;
	<ol>
		<li>Passage hiPSCs when colonies are between 0.8 -&#160;1.2 mm in diameter. Prior to passaging, check hiPSC colonies in a stereo-microscope and remove any areas of differentiation from the culture using the plastic edge of a 200 &#181;L tip.</li>
		<li>Aspirate spent media and add 10 mL hiPSC media (human ES culture media [Table of Materials] + 10 ng/mL human basic fibroblast growth factor [hbFGF]) supplemented with 10 &#956;M ROCK inhibitor.</li>
		<li>Hold the cell culture dish in one hand and roll a disposable cell passaging tool across the entire dish in one direction. Apply enough pressure so that the entire roller blade touches the culture dish and maintain a uniform pressure during rolling action.</li>
		<li>Rotate the culture dish 90&#176; and repeat step 1.2.3. View the plate in the microscope to visually confirm proper cutting of the colonies, which should appear checkered. Detach cut colonies by gentle mechanical flushing using a 200 &#956;L pipette.&#160; 
		NOTE: Detachment of cut colonies by mechanical flushing must be done immediately after cutting colonies with the roller, because after 3 min the cut colonies will start to reattach to the dish, and it will become difficult to detach colonies of homogeneous size by flushing.</li>
		<li>Transfer 350 -&#160;600 clumps of cut colonies onto a new 10 cm dish of MEF (plated 8 -&#160;36 h prior to hiPSC passaging) with 10 mL of fresh hiPSC media supplemented with 10 &#956;M ROCK inhibitor. Incubate at 37 &#176;C. 
		NOTE: 600 clumps represents approximately 1.0 x 106 MART-1 iPSC and will yield 0.5-1.0 x 106 DP cells on day 35. However, expected numbers will vary depending on the potency of the starting cell line and culture conditions.</li>
		<li>The following day, aspirate spent media and add 10 mL of fresh hiPSC media. Change hiPSC media every 1-2 days depending on hiPSC growth rate.</li>
	</ol>
	</li>
</ol>
2. Preparation of OP9/DLL1 Cells for Co-culture with hiPSCs
<ol>
	<li>Culture OP9/DLL1 cells in OP9 media [&#945;-minimum essential medium (&#945;-MEM) + 20% fetal bovine serum (FBS) + 1x penicillin-streptomycin] at 37 &#176;C. When OP9/DLL1 cells reach confluency, aspirate media and wash once with 5 mL of 1x magnesium, calcium, and phenol red-free phosphate buffered saline (PBS).</li>
	<li>Aspirate PBS and add 2 mL of 0.05% Trypsin-EDTA.&#160; Incubate for 5 min at 37 &#176;C. Then, add 4 mL of OP9 media and mechanically dissociate the cell layer by pipetting to make a single-cell suspension.</li>
	<li>Transfer the cell suspension into a 50 mL conical tube through a 100 &#956;m cell strainer to avoid cell clumps. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 12 mL of OP9 media.&#160;</li>
	<li>Add 8 mL of OP9 media to each of six new 10 cm cell culture Petri dishes. Plate 2 mL of OP9/DLL1 cell suspension from step 2.3 onto each new 10 cm dish. Rock the dish side-to-side then front-to-back to ensure even distribution of OP9/DLL1 over the dish.</li>
	<li>Incubate at 37 &#176;C. Repeat passage every 2 -&#160;3 days when cells reach confluency. 
	NOTE: It is important to make enough frozen stock of OP9/DLL1 cells and thaw a new stock every 4-6 weeks.</li>
</ol>
3. In Vitro Differentiation of hiPSCs into CD8&#945;&#946;+ Single Positive (SP) T Cells
<ol>
	<li>Prepare gelatinized OP9/DLL1 dishes one week prior to co-culture with hiPSCs. To prepare 0.1% gelatin solution, add 5 mL of room temperature (RT) tissue-grade stock gelatin solution to 500 mL of PBS.
	<ol>
		<li>Coat 3 new 10 cm cell culture Petri dishes by adding 4 mL per dish of 0.1% gelatin.&#160; Incubate 30 min at 37 &#176;C.&#160;</li>
		<li>Aspirate gelatin and add 8 mL of OP9 media to each dish. Passage one confluent dish of OP9/DLL1 (as done in section 2 above) to three gelatin pre-coated dishes.</li>
	</ol>
	</li>
	<li>After 4 days, add 10 mL of OP9 media to each 10 cm dish of OP9/DLL1 on gelatin, for a total of 20 mL of media per dish.</li>
	<li>After 7 -&#160;8 days, begin the hiPSC co-culture on OP9/DLL1 confluent dishes (differentiation day 0).
	<ol>
		<li>Aspirate spent media from confluent 10 cm dish of hiPSCs on MEF. Add 10 mL of OP9 media. Cut and detach hiPSC colonies using a disposable cell passaging tool as done in steps 1.2.3 and 1.2.4.</li>
		<li>Transfer 350 -&#160;600 clumps of cut colonies onto a 10 cm pre-gelatinized OP9/DLL1 dish (step 3.1) with 10 mL of fresh OP9 media using a 200 &#956;L pipette. Rock the culture dish side-to-side then front-to-back to ensure even distribution of colonies. 
		NOTE: Alternatively, pre-formed hiPSC embryoid bodies (EBs) or small clump suspension may be used.&#160; However, the use of a disposable cell passaging tool or EB formation system is preferred to produce hiPSC clumps of uniform size.</li>
	</ol>
	</li>
	<li>On day 1, aspirate spent media and replace with 20 mL of fresh OP9 media. hiPSC clumps co-cultured on OP9/DLL1 for 1 day will appear as small round monolayer colonies (Figure 1).</li>
	<li>On day 5, aspirate 10 mL of spent media and add 10 mL of fresh OP9 media. hiPSC colonies will begin to differentiate into primitive mesoderm, which is characterized by a multilayered dark center.</li>
	<li>On day 9, aspirate 10 mL of spent media and add 10 mL of fresh OP9 media. At this point, multilayered center structures will evolve into dome-like shapes, and a peripheral network-like area will start to become evident.</li>
	<li>On day 13, harvest hematopoietic progenitor cells (HPCs) (Figure 1). hiPSC-derived structures on day 13 are characterized by a dark central organoid surrounded by a network of dome-like areas, representative of hematopoietic zones (HZs) previously reported to enclose human embryonic stem cell-derived hematopoietic progenitors21. 
	NOTE: The presence of the dome-like structures indicates a successful process even in the absence of dark centers. The inability to produce HPCs may be due to poor quality of OP9/DLL1, quality of FBS lot, confluency of iPSC clumps seeded onto OP9/DLL1 (350-600 clumps is optimal), and/or variations in potency of iPSC lines to produce hematopoietic precursors.
	<ol>
		<li>Aspirate spent media and wash 1x with 5 mL of 1x phenol red-free Hanks&#39;&#160;balanced salt solution modified with calcium and magnesium (HBSS).&#160;</li>
		<li>Aspirate HBSS and add 250 &#956;L of 5000 Units/mL collagenase IV in 10 mL of HBSS. Incubate at 37 &#176;C for 45 min. Aspirate HBSS with collagenase IV and wash once with 5 mL of PBS.</li>
		<li>Aspirate PBS and add 5 mL of 0.25% Trypsin-EDTA. Incubate at 37 &#176;C for 20 min. Then, add 4 mL of OP9 media and dissociate the cell layer by pipetting to make a single-cell suspension.</li>
		<li>Transfer the cell suspension into a 50 mL conical tube through a 100 &#956;m cell strainer.&#160; Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 10 mL of OP9 media.&#160;</li>
		<li>Plate cell suspension onto a new gelatinized 10 cm cell-culture Petri dish (see steps 3.1.1 and 3.1.2). Incubate at 37 &#176;C for 45 min. Then, collect non-adherent cells by gentle pipetting.</li>
		<li>Transfer collected cell suspension into a 50 mL conical tube through a 100 &#956;m cell strainer.&#160; Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 10 mL of differentiation media [OP9 media with 5 ng/mL human stem cell factor (hSCF), 5 ng/mL human Flt3 ligand (hFLT3L), and 5 ng/mL human interleukin 7 (hIL-7)].</li>
		<li>Plate the cell suspension onto a new 10 cm OP9/DLL1 confluent dish.&#160;</li>
	</ol>
	</li>
	<li>On day 16, passage the cells.&#160;
	<ol>
		<li>Mechanically detach non-adherent cells by gentle pipetting and filter through a 100 &#956;m cell strainer. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 10 mL differentiation media.</li>
		<li>Plate the cell suspension onto a new 10 cm OP9/DLL1 confluent dish.&#160;</li>
	</ol>
	</li>
	<li>Continue passaging non-adherent cells every 5-7 days thereafter by repeating step 3.8.</li>
	<li>On day 35, enrich CD4+CD8+ double positive (DP) population and stimulate to produce CD8&#945;&#946;+ SP T cells (Figure 2).
	<ol>
		<li>Mechanically detach non-adherent cells by gentle pipetting and filter through a 100 &#956;m cell strainer to remove cell clumps. Enrich CD4+ cell population by CD4 magnetic bead isolation according to the manufacturer&#8217;s protocol. 
		NOTE: The rationale for using CD4 magnetic beads is to remove CD4-CD8- DN cells from the culture, as these have been demonstrated to cause direct killing of CD4+CD8+ DP cells after stimulation22.</li>
		<li>Count live CD4 enriched cells using a Neubauer hemocytometer and Trypan blue dye.&#160; Suspend in OP9 media at total concentration 0.5 x 106 cells/mL. Aliquot 1 mL of the cell suspension (0.5 x 106 cells) into each well of a tissue culture flat bottom 24 well plate of confluent OP9/DLL1.</li>
		<li>Add 100 IU human interleukin 2 (hIL-2), 5 ng/mL hIL-7, 500 ng/mL anti-human CD3 antibody, and 2 &#956;g/mL anti-human CD28 antibody, then culture at 37 &#176;C.</li>
		<li>On day 4 -&#160;7 after stimulation, collect cells for molecular analysis (Figure 3) or co-culture with peptide-pulsed antigen presenting cells (APCs).</li>
	</ol>
	</li>
</ol>
4. Measuring Antigen Specificity of hiPSC Derived CD8&#945;&#946;+ SP T Cells
NOTE: The type of APCs to be used for this experiement is dependent on the MHC restriction of hiPSC-derived T cells. Here, T2 cell line is used, which is a hybrid of T and B lymphoblastoid cell lines. T2 cells express HLA-A*02:0123, which is recognized by JKF6 cells from which MART1-iPSC was derived18. This T2 cell line can be expanded in RPMI 1640 + 20% FBS + 1x penicillin-streptomycin and is passaged when cells reach a density of 5 x 105 cells/mL.
<ol>
	<li>Count live HLA-A*02:01+ T-B hybrid lymphoblastoid T2 cells using a Neubauer hemocytometer and Trypan blue dye. Incubate APCs in 24 well tissue culture plate with 1 &#956;g/mL MART-1 peptide for 2 h at 37 &#176;C. &#160; 
	NOTE: Optimal peptide concentration is variable, depending on cell line and antigen specificity.</li>
	<li>Collect APCs and wash 2x with 10 mL of PBS to remove any extra peptide.</li>
	<li>Count APCs and suspend at 2 &#8211; 5 x 105 cells/mL in OP9 media with 100 IU IL-2 and 5 ng/mL IL-7. Aliquot 100 &#956;L of cell suspension (2-5 x 104 cells) into each well of an ultra-low attachment U bottom 96 well plate or directly into a pre-coated ELISpot plate.</li>
	<li>Sort hiPSC-derived CD8&#945;&#946;+ SP T cells (1 week after anti-human CD3/CD28 antibody stimulation) using a cell sorter and suspend at 1 x 106 cells/mL in OP9 media with 100 IU IL-2 and 5 ng/mL IL-7. Aliquot 100 &#956;L of cell suspension (1 x 105 cells) into each well of APCs and culture for 16 -&#160;20 h at 37 &#176;C.</li>
	<li>After 16 -&#160;20 h, analyze the cytokine secretion profile by ELISpot assay per the manufacturer&#39;s protocol (Figure 4).</li>
</ol>

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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=CD8alpha/alpha+homodimers+fail+to+function+as+co-receptor+for+a+CD8-dependent+TCR.">CD8alpha/alpha homodimers fail to function as co-receptor for a CD8-dependent TCR.</a> European Journal of Immunology. 37 (6), 1634-1641 (2007).</li><li>Themeli, M., Riviere, I., Sadelain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+cell+sources+for+T+cell+engineering+and+adoptive+immunotherapy.">New cell sources for T cell engineering and adoptive immunotherapy.</a> Cell Stem Cell. 16 (4), 357-366 (2015).</li><li>Minagawa, A., 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=Enhancing+T+Cell+Receptor+Stability+in+Rejuvenated+iPSC-Derived+T+Cells+Improves+Their+Use+in+Cancer+Immunotherapy.">Enhancing T Cell Receptor Stability in Rejuvenated iPSC-Derived T Cells Improves Their Use in Cancer Immunotherapy.</a> Cell Stem Cell. 23 (6), 850-858 (2018).</li><li>Montel-Hagen, A., 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=Organoid-Induced+Differentiation+of+Conventional+T+Cells+from+Human+Pluripotent+Stem+Cells.">Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells.</a> Cell Stem Cell. 24 (3), 376-389 (2019).</li><li>Takada, K., Kondo, K., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Peptides+That+Promote+Positive+Selection+in+the+Thymus.">Generation of Peptides That Promote Positive Selection in the Thymus.</a> Journal of Immunology. 198 (6), 2215-2222 (2017).</li><li>Takada, K., 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=TCR+affinity+for+thymoproteasome-dependent+positively+selecting+peptides+conditions+antigen+responsiveness+in+CD8(+)+T+cells.">TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.</a> Nature Immunology. 16 (10), 1069-1076 (2015).</li><li>Vizcardo, 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=A+Three-dimensional+Thymic+Culture+System+to+Generate+Murine+Induced+Pluripotent+Stem+Cell-derived+Tumor+Antigen-specific+Thymic+Emigrants.">A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants.</a> JoVE. , e58672 (2019).</li></ol>

The authors have no disclosures.

The co-culture of OP9 murine stromal cells is a well-established system for in vitro generation of lymphocytes (i.e., NK, B, and T cells) from HSPCs and pluripotent stem cells. Notch signaling is required to induce T lineage commitment and can be accomplished by the ectopic expression of Notch ligand DLL1 or DLL4, which have comparable efficacy for T cell generation1. Therefore, the OP9/DLL1 co-culture system has become a widely used method for producing T cells in vitro.&#160; Furthermore, this method is applicable for use with several types and sources of human cells, including cord blood, bone marrow HSPCs and ESCs.&#160; However, the generation of T cells from these sources is limited either by insufficient retrieval of source cells or by inefficient differentiation to T cells1. Additionally, a T cell product with a single TCR recombination cannot be generated from these open repertoire sources. By using regenerative medicine techniques, namely induced pluripotent stem cell (iPSC) technology, it may be possible to produce massive numbers of antigen-specific T cells for use in cell-based therapeutics15.&#160;
hiPSCs are similar to pluripotent ESCs in their capacity for self-renewal, limitless expansion, and potential to differentiate to any type of somatic cell in the body; however, they lack the ethical concerns surrounding the use of products of embryonic origin for clinical applications. Moreover, hiPSCs can be produced from any somatic cell, allowing the development of cell products for personalized medicine. In previous reports, hiPSCs have been produced from human T cells using whole peripheral mononuclear cells, CD3+ cells, or isolated cytotoxic T lymphocytes (CTLs) as a source18,19,22,26. When hiPSCs are generated from a T cell source (T-iPSCs), the original TCR gene rearrangement is inherited. Therefore, patient T-iPSC derived T cells may provide a model for personalized ACT treatment by targeting a patient&#8217;s distinct cancer antigens.
The differentiation of human pluripotent stem cells into T lineage cells is divided into two steps: the generation of hematopoietic progenitor cells (HPCs)27 and their further differentiation into T lineage cells21. Both steps can be accomplished using the OP9/DLL1 co-culture system. Importantly, the quality of OP9/DLL1 feeder cells is critical to the success of T cell differentiation. Since OP9/DLL1 cells are not an immortalized homogenous cell line, the quality of the FBS and culture conditions are critical to maintaining their expansion without losing the ability to support hiPSC differentiation. Therefore, it is recommended to pre-evaluate the lot of FBS and passage consistently when cell-to-cell cytoplasmic contact begins to occur, in order to prevent cell differentiation and senescence. One point to take into consideration is that cell-to-cell contact can appear indistinguishable from the background depending on the phase contrast and magnification of the microscope. In our experience, most OP9/DLL1 dishes will appear to be 80% confluent when ready to passage.
It has been shown that the redifferentiated T lineage cells generated from T-iPSCs by OP9/DLL1 co-culture can produce CD8+ SP T cells upon stimulation18,19. However, regenerated CD8+ SP T cells acquire the innate-like CD8&#945;&#945; homodimer22,28, which is an ineffective co-receptor for TCR signaling29. &#160;Additionally, these regenerated CD8+ SP T cells have shown strong TCR-independent cytotoxicity, making these cells unfavorable for clinical use30. This protocol describes a recent method involving stimulation of purified CD4+CD8+ DP cells to generate CD8&#945;&#946;+ SP T cells with a more conventional phenotype and improved antigen-specific cytotoxicity22. Although the loss of antigen specificity due to secondary TCR&#945; allelic rearrangement occurs in the DP stage after prolonged long-term culture, this can be overcome by genome editing in T-iPSCs31.&#160; In our experience, hiPSC-derived DP cells start to appear on day 30 -&#160;35 of culture, and these newly generated DP cells have not yet undergone secondary TCR&#945; rearrangement. Therefore, most DP cells on day 35 retain antigen specificity and can be used to generate antigen-specific CD8&#945;&#946;+ SP T cells. &#160;
Prior to human anti-CD3 and anti-CD28 stimulation on day 35, CD4-CD8- DN cells must be removed from the culture, as these have been demonstrated to cause direct killing of CD4+CD8+ DP cells after stimulation22. Using CD4 magnetic bead enrichment (step 3.10) will enrich for both DP and CD4+CD8- intermediate single positive (ISP) cells1, which we have been shown to have no negative effects22. Alternatively, fluorescence-activated cell sorting by flow cytometry can be performed to isolate DP cells. However, magnetic bead separation is preferred as it avoids the mechanical stress induced by flow cytometry.
The generation of CD8&#945;&#946;+ SP T cells from human pluripotent stem cells without activation-mediated agonist selection has subsequently been demonstrated by the use of 3D murine stromal cell culture32. However, physiological positive selection is dependent on the interaction of TCR with self-peptide-MHC complexes, which are uniquely processed and presented by thymic cortical epithelial cells33. Furthermore, TCR affinity for selection peptides has been shown to determine the subsequent functional capabilities of mature CD8&#945;&#946;+ SP T cells34.&#160; Currently, there is no evidence to suggest that a Notch stromal cell-based co-culture system can provide the defined selection peptide and MHC complex required for physiological positive selection.
It has been previously reported in a murine model that T lineage cells generated from tumor antigen-specific T cell-derived hiPSCs using OP9/DLL1 alone fail to experience conventional maturation. However, iPSC-derived immature T cells generated by the OP9/DLL1 system can mature into na&#239;ve-like T cells by further physiological thymic education in a 3D culture system 28,35. Therefore, the protocol presented here to produce iPSC-derived immature T cells generated by the OP9/DLL1 system is vital for further attempts to generate real human tumor antigen-specific post-thymic T cells capable of long-term persistence in vivo with efficiency to treat established vascularized tumors.

In addition to physiological advantages, T cells have many potential therapeutic applications. The generation and expansion of T cells in vitro can be used for disease modeling and therapeutic validation as well as a source of treatment for hereditary and acquired immunodeficiency states (i.e., viral immunodeficiencies and lymphodepletion secondary to chemotherapy or transplantation) and for the eradication of cancer. This latter quality has led to the development of adoptive T cell transfer (ACT) for the treatment of patients with advanced cancer1.&#160;
ACT consists of resecting a patient&#8217;s tumor, extracting tumor-infiltrating lymphocytes (TILs), expanding TILs ex vivo, then reinfusing the expanded cells into the patient2. It has been shown to be an effective treatment modality for some patients with metastatic cancer. &#160;Unfortunately, not all patients respond to this therapy. Previous reports have shown that the differentiation state of transferred cells3,4,5,6,7,8,9, use of large numbers of highly enriched cancer antigen-specific T cells10, and persistence of T cells after transfer11,12 are all correlated with more durable responses13,14. Therefore, when ACT fails to elicit an anti-tumor response, it may in part be due to a low yield of cancer antigen-specific T cells, inefficient ex vivo expansion leading to the exhaustion and loss of reactive clones, or lack of persistence after transfer4. It has been postulated that these obstacles may be overcome by the generation of large numbers of less differentiated cancer antigen-specific T cells in vitro15,16.&#160;
Hematopoietic stem/progenitor cells (HSPCs) are a conventional source for in vitro T cell generation, although this method is limited by the small number of cells able to be recovered from a single donor1. Embryonic stem cells (ESCs) have also been shown to produce T cells but with low yield17, making it inefficient for clinical applications. Furthermore, since T lineage cells experience stochastic genetic recombination of their T cell receptors (TCRs) in early developmental stages, it is not possible to use HSPCs or ESCs to generate a pure population of antigen-specific T cells without further genomic modifications like TCR gene transduction.
One approach to overcome these caveats is to reprogram TILs to human induced pluripotent stem cells (hiPSCs), which may provide a limitless source for in vitro T cell generation. It has been shown that cancer antigen-specific TILs can be reprogrammed into hiPSCs and re-differentiated to T cell lineage, which retains the same T cell receptor (TCR) gene rearrangement as the original T cell18,19.&#160; This detail is important for ACT because individual patient tumors have unique mutational profiles, and very few cancer antigens have been shown to be shared among patients20. Therefore, using cancer antigen-specific TILs as a source for in vitro generation of hiPSC-derived T cells may provide a new strategy for the personalized treatment of patients with metastatic cancer.
Presented here in detail is a protocol for differentiating hiPSC-derived T lineage cells into functional antigen-specific CD8&#945;&#946;+ single positive (SP) T cells using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell differentiation of hiPSCs, hematopoietic progenitors, and embryonic stem cells, as well as their further applications in regenerative medicine and cell-based therapies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10 cm dish</td>
			<td >Corning, Inc.&#160;</td>
			<td >353003</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-CD3, human</td>
			<td >BD Biosciences</td>
			<td>Cat# 561812, RRID:AB_1089628</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34, human</td>
			<td >BD Biosciences</td>
			<td >Cat# 348791, RRID:AB_400381</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD4, human</td>
			<td >Biolegend</td>
			<td>Cat# 344612, RRID:AB_2028479</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD43, human</td>
			<td >BD Biosciences</td>
			<td >Cat# 560198, RRID:AB_1645460</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD7, human</td>
			<td >BD Biosciences</td>
			<td>Cat# 555361, RRID:AB_395764</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-CD8a, human</td>
			<td >BD Biosciences</td>
			<td>Cat# 555369, RRID:AB_398595</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-CD8b, human</td>
			<td >BD Biosciences</td>
			<td >Cat# 641057, RRID:AB_1645747</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-TCRb, human</td>
			<td >BD Biosciences</td>
			<td >Cat# 555548, RRID:AB_395932</td>
			<td ></td>
		</tr>
		<tr >
			<td >CD28 human monoclonal antibody (15E8), pure functional grade</td>
			<td >Miltenyl Biotec</td>
			<td>130-093-375</td>
			<td ></td>
		</tr>
		<tr >
			<td >CD3 human monoclonal antibody (OKT3), pure functional grade</td>
			<td >Miltenyl Biotec</td>
			<td>130-093-387</td>
			<td ></td>
		</tr>
		<tr >
			<td >CD4 Microbeads, human</td>
			<td >Miltenyl Biotec</td>
			<td >130-045-101</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cell strainer 100 um</td>
			<td >Fisher Scientific</td>
			<td >22-363-549</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td >Gemini</td>
			<td >100-500</td>
			<td></td>
		</tr>
		<tr >
			<td >Flt-3 ligand</td>
			<td >R&#38;D Systems</td>
			<td >427-FL</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%&#160;</td>
			<td>SIGMA-Aldritch</td>
			<td>G1393-100ML</td>
			<td></td>
		</tr>
		<tr >
			<td >GlutaMAX (100X)</td>
			<td >Thermo Fisher Scientific</td>
			<td >35050-061</td>
			<td >L-Glutamine supplement</td>
		</tr>
		<tr >
			<td >HBSS Mg+Ca+ Phenol-Red Free</td>
			<td >Gibco</td>
			<td >14025-092</td>
			<td></td>
		</tr>
		<tr >
			<td >Interleukin-2</td>
			<td >R&#38;D Systems</td>
			<td >202-IL</td>
			<td></td>
		</tr>
		<tr >
			<td >Interleukin-7</td>
			<td >R&#38;D Systems</td>
			<td >407-ML</td>
			<td></td>
		</tr>
		<tr >
			<td >iTAG MHC Tetramer HLA-A*0201 Mart1 Tetramer -ELAGIGILTV</td>
			<td >MBL</td>
			<td >Cat#TB-0009-2</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mart1-hiPSC</td>
			<td >Vizcardo et al., Cell Stem Cell 2013</td>
			<td >RIKEN-IMS</td>
			<td></td>
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		<tr >
			<td >Melan-A, MART 1 (26-35)</td>
			<td >InnoPep</td>
			<td >3146-0100</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM Non-Essential Amino Acids Solution</td>
			<td >Gibco</td>
			<td >11140050</td>
			<td></td>
		</tr>
		<tr >
			<td >&#945;MEM powder</td>
			<td >Gibco</td>
			<td >61100061</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse Embryonic Fibroblasts (MEF)</td>
			<td >Thermo Fisher Scientific</td>
			<td>C57BL/6 MEF MITC-TREATED 4M EACH; A34962</td>
			<td></td>
		</tr>
		<tr >
			<td >OP9/N-DLL1</td>
			<td >Riken Bioresource center</td>
			<td >Cat# RCB2927; RRID:CVCL_B220</td>
			<td >OP9/DLL1</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin</td>
			<td >Thermo Fisher Scientific</td>
			<td >15140-122</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate buffered saline pH 7.4 (1x)</td>
			<td >Thermo Fisher Scientific</td>
			<td >10010-023</td>
			<td></td>
		</tr>
		<tr >
			<td >Primate ES Cell Medium</td>
			<td >Reprocell</td>
			<td >RCHEMD001</td>
			<td >Human ESC Culture Media</td>
		</tr>
		<tr >
			<td >Rhok inhibitor (Y-27632 dihydrochloride)</td>
			<td >Tocris</td>
			<td >1254</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI 1640</td>
			<td >Gibco</td>
			<td >11875093</td>
			<td></td>
		</tr>
		<tr >
			<td >Stem Cell Factor (SCF)</td>
			<td >R&#38;D Systems</td>
			<td >455-MC</td>
			<td></td>
		</tr>
		<tr >
			<td >StemPro</td>
			<td >EZPassage</td>
			<td >23181-010</td>
			<td></td>
		</tr>
		<tr >
			<td >T2-tumor</td>
			<td >ATCC</td>
			<td>T2 (174 x CEM.T2) (ATCC&#174; CRL-1992&#8482;)&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin-EDTA (0.05%), phenol red&#160;</td>
			<td >Thermo Fisher Scientific</td>
			<td >25300-062</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin-EDTA (0.25%), phenol red&#160;</td>
			<td >Thermo Fisher Scientific</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >U Bottom 96 well plate</td>
			<td >Corning, Inc.&#160;</td>
			<td >3799</td>
			<td></td>
		</tr>
	</tbody>
</table>

using human induced pluripotent stem cells for the generation of tumor antigen-specific t cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

After 13 days hiPSCs co-culturing with OP9/DLL1, CD34+CD43+ hematopoietic progenitor cells appeared (Figure 1). After an additional 22 days of culture on non-gelatinized OP9/DLL1 in the presence of hSCF, hFLT3L, and hIL-7, hematopoietic progenitors differentiated into CD3+CD7+CD4+CD8+ double-positive (DP) T lineage cells, the majority of which expressed TCR specific to the MART-1 epitope (tetramer) (Figure 2).
It has previously been shown that CD8+ SP T cells can be induced from CD4+CD8+ DP T cells by TCR signaling via agonist peptide or antibody-driven TCR stimulation24,25. Therefore, on day 35 of culture, hiPSC-derived CD4+CD8+ DP T cells were stimulated with anti-human CD3 and anti-human CD28 antibodies in the presence of hIL-7 and hIL-2.&#160; Four days after stimulation, the number of CD3+CD8&#945;&#946;+ SP cells increased dramatically and remained specific for the MART-1 epitope, confirming the preservation of their inherited antigen-specificity (Figure 3).&#160;
To determine the functional properties of hiPSC-derived CD8&#945;&#946;+ SP T cells, antigen-dependent activation and secretion of interferon gamma (IFN-&#947;) was analyzed. After stimulation with anti-human CD3 and anti-human CD28 antibodies for 1 week, hiPSC-derived CD8&#945;&#946;+ SP T cells were isolated using a cell sorter and co-cultured with T2 cell line expressing HLA-A*02:01 with or without cognate MART1 peptide for 16 -&#160;20 h. The ELISpot assay revealed that hiPSC-derived CD8&#945;&#946;+ SP T cells secrete higher amounts of IFN-&#947; compared with CD8&#945;&#945;+ SP T cells, when cultured in the presence of MART-1 peptide. IFN-&#947; expression was null for T cells and APCs alone, demonstrating that human T-iPSC-derived T cells are antigen-specific and functional (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59997/59997fig1.jpg" /> 
Figure 1: Generation of hiPSC-derived hematopoietic progenitor cells. (A) Schematic overview of the differentiation of hiPSCs to hematopoietic lineage using OP9/DLL1 co-culture. (B) Appearance of hiPSC-derived structures on days 1 (top left), 3 (top right), 7 (bottom left), and 13 (bottom right). Scale bars = 100 &#181;m. (C) Flow cytometric analysis of hiPSC derived CD34+CD43+ hematopoietic progenitor cells on day 13. Data are representative of six independent experiments (n = 1 to 2). <a href="https://www.jove.com/files/ftp_upload/59997/59997fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59997/59997fig2.jpg" /> 
Figure 2: hiPSC differentiation into Mart1+ CD4+CD8&#945;&#946;+ DP T cells. (A) Schematic overview of the differentiation of hiPSC-derived hematopoietic lineage to immature T cells using OP9/DLL1 co-culture. (B) Flow cytometric analysis of CD4 vs. CD8&#945;, CD3 vs. CD8&#946;, and MART-1 tetramer expression in hiPSC-derived T cells on day 35. Gated on lymphocytes, single cells, PI negative. Data are representative of three independent experiments (n = 3 -&#160;8). <a href="https://www.jove.com/files/ftp_upload/59997/59997fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59997/59997fig3.jpg" /> 
Figure 3: Induction of CD8&#945;&#946;+ SP T cell phenotype. Flow cytometric analysis of CD4- hiPSC-derived T cells 4 days after human anti-CD3 and human anti-CD28 antibody-driven stimulation. Gated on lymphocytes, single cells, PI negative (n = 4). &#160; <a href="https://www.jove.com/files/ftp_upload/59997/59997fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59997/59997fig4.jpg" /> 
Figure 4: Antigen specificity of hiPSC-derived CD8&#945;&#946;+ SP T cells. IFN-&#947; secretion by ELISpot assay of hiPSC-derived CD8&#945;&#946;+ SP, CD8&#945;&#945;+ SP, and bulk T cells after 20 h co-culture with or without T2 cells pulsed (or not) with MART-1 peptide. <a href="https://www.jove.com/files/ftp_upload/59997/59997fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells at JoVE.com

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

using-human-induced-pluripotent-stem-cells-for-generation-tumor

Research

JoVE Journal

Cancer Research

10.2K Views.  National Cancer Institute, NIH. This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8αβ+ single positive T cells using OP9/DLL1 co-culture system.

Video: Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

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.

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.

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

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...