This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Projektnummer 360372040 - SFB 1335 and Projektnummer 395357507 - SFB 1371 (to H.P.), a Mechtild Harf Research Grant from the DKMS Foundation for Giving Life (to H.P.) a Young Investigator Award by the Melanoma Research Alliance (to S.H.), a scholarship by the Else-Kr&#246;ner-Fresenius-Stiftung (to F.S.), a Seed Fund by the Technical University Munich (to S.H.) and a research grant by the Wilhelm Sander Foundation (2021.041.1, to S.H.). H. P. is supported by the EMBO Young Investigator Program.
AUTHOR CONTRIBUTIONS:
F.S., H.P., and S.H. designed the research, analyzed, and interpreted the results. F.S. and S.H. wrote the manuscript. H.P. and S.H. guided the study.

At the onset of experiments, mice were at least 6 weeks of age and were maintained under specific pathogen-free conditions. The present protocol complies with the Institutional ethical standards and prevailing local regulations. Animal studies were approved by the local regulatory agency (Regierung von Oberbayern, Munich, Germany). Possible sex-related biases were not investigated in these studies.
1. Generation and isolation of EVs derived from tumor cells after chemotherapy exposure
<ol>
	<li>Culture murine B16 melanoma cells expressing ovalbumin (B16-OVA) in DMEM (containing 4 mM L-glutamine and 4.5 g/L D-glucose) supplemented with FCS (10% v/v), penicillin (100 Units/mL), and streptomycin (100 &#956;g/mL) at 37 &#176;C, until cells grow steadily and are approximately 90% confluent. 
	NOTE: Perform this section of the protocol entirely under sterile conditions using a cell culture hood. For analysis of other cancer entities, cell lines expressing a potent antigen are preferable to assess antigen-specific T cell responses, as they allow for specific ex vivo antigen restimulation.</li>
	<li>To prepare the cell culture media required for EV generation, deplete bovine EVs within FCS by ultracentrifugation at 100,000 x g for 24 h at 4 &#176;C, and then discard the pellet. Alternatively, choose a commercial preparation low in animal EVs beforehand.</li>
	<li>Harvest B16-OVA cells and wash them twice in PBS, and then seed at a concentration of 400,000 cells/mL in EV-depleted media. 
	NOTE: Adapt the cell concentration to the growth dynamic of the cell line under investigation so that cells do not overgrow.</li>
	<li>Treat B16-OVA cells by adding 30 &#956;g of oxaliplatin per mL and incubate for 24 h at 37 &#176;C. Leave control conditions untreated. At the first use of a genotoxic substance, titrate the desired cytotoxic efficacy using cell-viability assays such as trypan blue exclusion18. 
	NOTE: This assay may also evaluate EVs generated in cell cultures treated with other immune-modulating substances or ionizing irradiation besides chemotherapeutics. 
	CAUTION: Oxaliplatin causes skin and severe eye irritation and may cause an allergic skin reaction and respiratory irritation. Oxaliplatin is suspected of causing cancer. As a precaution, use personal protective equipment, including adequate gloves, goggles, masks, and clothing, cleaned before reuse. Avoid inhalation and wash hands thoroughly after handling. Avoid release into the environment and dispose oxaliplatin according to prevailing regulations. Obtain detailed information from the safety data sheet.</li>
	<li>Collect cell culture supernatant. Centrifuge first at 400 x g for 5 min at 4 &#176;C, and then at 2,000 x g for 30 min at 4 &#176;C, each time discarding the pellet. Finally, filter through a 220 nm PVDF membrane. Use a fresh tube for each step to remove any cell debris. 
	NOTE: At this stage, the EV-containing supernatant may be stored at 4 &#176;C for a day before resuming the protocol. However, it is strongly recommended to adhere to the described schedule with immediate EV purification.</li>
	<li>Mix 1 mL of supernatant with 0.5 mL of a specific commercially available exosome isolation reagent (see Table of Materials) in a V-shaped 1.5 mL tube. Thoroughly pipette up and down or vortex to create a homogenous solution. Incubate overnight at 4 &#176;C.</li>
	<li>Centrifuge at 10,000 x g for 60 min at 4 &#176;C. Carefully discard the supernatant. Remove the remaining drops by tapping the 1.5 mL tube upside down on a paper towel and by aspiration through a pipette with a fine tip without touching the EV-pellet at the bottom.
	<ol>
		<li>Thoroughly remove all fluids to prevent uncontrolled dilution of the EV-pellet. Also, execute these tasks quickly to prevent the pellet from drying out.</li>
	</ol>
	</li>
	<li>Resuspend the EVs in cold PBS by pipetting up and down without scratching the pellet from the tube&#39;s wall with the tip. Now, transfer the suspension step by step from the first to the last tube to pool the EVs. 
	NOTE: Use a volume of PBS that equals 5 &#956;L multiplied by the number of tubes. 5 &#956;L of the final suspension contains the isolated EVs released from 400,000 cells under chemotherapy or at a steady state.</li>
	<li>Preferably, use EVs directly. If this is not possible, store EV suspensions at -80 &#176;C in siliconized vessels for up to 28 days until application. 
	NOTE: The EVs described here and in several other publications do not lose their respective biological function when stored at -80 &#176;C for that time period19.</li>
	<li>Quantify and characterize EV isolates according to the MISEV2018 guidelines20. 
	NOTE: Possible methods for quantification include nanoparticle tracking analysis (NTA)21 and the detection of EV&#39;s membrane-bound proteins22. Possible approaches to further characterize EVs include electron microscopy23 and western blot20.</li>
</ol>
2. Immunization of mice with EVs
<ol>
	<li>Plan the in vivo experiment with C57BL/6 mice (or other syngeneic mice corresponding to the tumor cell line), including treatment groups receiving EVs derived from treated cells, untreated cells, and PBS (vehicle), respectively. 
	NOTE: Preferably, use mice at the age of 6-8 weeks to prevent physiological senescence from diminishing the immune response24.</li>
	<li>Mix 5 &#956;L of EV-suspension with 55 &#956;L cold PBS for each mouse within the respective treatment group to immunize it with EVs isolated from 4.0 x 105 B16-OVA cells. 
	NOTE: This amount of EVs corresponds to approximately 2 x 109 particles measured by nanoparticle tracking analysis (data not shown). OVA protein mixed with an adjuvant (e.g., LPS) can be applied as a potent vaccine positive control.
	<ol>
		<li>Fill syringes (needle size 26-30 G) with 60 &#956;L of the diluted EVs or PBS, respectively and put immediately on ice. 
		NOTE: In the protocol, the amount of injected EVs is normalized to the number of EV-releasing tumor cells to experimentally consider both qualitative and quantitative effects of oxaliplatin on tumor cell EV biogenesis. For some readers, normalization to a specific concentration of produced EVs may better fit their experimental setup depending on their scientific question.</li>
	</ol>
	</li>
	<li>Inoculate EVs or PBS subcutaneously into the medial aspect of the mice&#39;s thigh&#160;and repeat the immunization after 7 days. Fourteen days after the first treatment, sacrifice mice, e.g., by cervical dislocation to analyze the immune response. 
	&#8203;NOTE: Alternative subcutaneous injection routes may be used, according to the local standards.</li>
</ol>
3. Flow cytometry analysis of splenic T cells
<ol>
	<li>Prepare and cool complete RPMI (cRPMI), supplementing RPMI-1640 with FCS (10% v/v), penicillin (100 Units/mL), streptomycin (100 &#956;g/mL), L-glutamine (2 mM), and &#946;-mercaptoethanol (50 &#956;M).</li>
	<li>Resect the spleen from the opened abdominal cavity. Mash the spleen with a moistened 100 &#956;m cell strainer and the plastic plunger of a syringe and flush the splenic cells into a 50 mL tube with 5-10 mL of cRPMI. Centrifuge at 400 x g for 5 min at 4 &#176;C and discard the supernatant. 
	NOTE: Keep cells on ice whenever possible. To analyze the local rather than the splenic immune response, resect the draining popliteal and inguinal lymph nodes, following the same protocol. In this case, skip the next step for the lysis of erythrocytes.</li>
	<li>To remove erythrocytes from the cell suspension, resuspend the pellet with 2 mL of red blood cell lysis buffer (see Table of Materials) and incubate for 5 min at room temperature. Then, stop the reaction by adding cRPMI. Centrifuge at 400 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>For seeding, resuspend the cell pellet in cRPMI and count the cells to place triplicates of 200,000 cells with 200 &#956;L cRPMI into each well, using a 96-well plate with a U-shaped bottom. Incubate for 48 h at 37 &#176;C. 
	NOTE: To address the antigen-specificity of activated T cells, add 1 &#956;g/mL soluble ovalbumin (or another tumor antigen corresponding to cell line under investigation) or leave without additional stimulus, respectively. Adding the immune-dominant peptide epitope SIINFEKL, instead of full-length ovalbumin, allows for a shorter incubation period. Besides flow cytometry, the mice&#39;s serum and the cell culture supernatant&#160;after 48 h of incubation can be analyzed for various cytokines.</li>
	<li>After 48 h, to enhance intracellular IFN-&#947; staining by, inter alia, blocking the Golgi-mediated secretion of proteins, add Brefeldin A (5 ng/mL), PMA (20 ng/mL), and Ionomycin (1 &#181;g/mL) to the cell culture. Incubate for 4 h at 37 &#176;C.</li>
	<li>Before staining surface biomarkers, transfer splenocytes to a 96-well plate with a V-shaped bottom and wash twice with PBS. Then, add fluorescent antibodies, compatible with the locally available flow cytometer, directed against surface biomarkers, CD3, CD8, and CD4 (see Table of Materials), diluted 1:400, plus a fixable viability dye, diluted 1:1,000 in PBS. Resuspend pelleted splenocytes in the staining solution and incubate for 30 min at 4 &#176;C, protected from light.</li>
	<li>For fixation and permeabilization of splenocytes, wash twice in FACS-buffer (PBS plus 3% v/v FCS), and then resuspend in 100 &#181;L of fixation/permeabilization buffer (see Table of Materials) per well. Incubate for 30 min at 4 &#176;C, protected from light.</li>
	<li>For staining of intracellular IFN-&#947;, wash splenocytes in fixation/permeabilization buffer and resuspend with fluorescent antibodies against IFN-&#947;, diluted 1:200 in the buffer. Incubate for at least 1 h (up to a maximum of 12 h) at 4 &#176;C, protected from light.</li>
	<li>Before measuring the samples by flow cytometry, wash splenocytes twice in fixation/permeabilization buffer and resuspend in FACS-buffer. Analyze the activation of cytotoxic T cells according to the gating strategy displayed in Figure 2.
	<ol>
		<li>First, to detect single cells, blot FSC-H against FSC-A. Then, to detect lymphoid cells, blot SSC against FSC-A. Subsequently, select living CD3+, CD4-, CD8+ cells and determine their IFN-&#947;-producing subset to quantify the activation of cytotoxic T cells in the spleen. 
		NOTE: Include a Fluorescence-minus-one (FMO) stain with all fluorochromes except the fluorochrome targeted against IFN-&#947; as negative technical control.</li>
	</ol>
	</li>
</ol>

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The authors declare that there is no conflict of interest.

This protocol provides an immunological in vivo assessment of EVs derived from melanoma cells under chemotherapy-induced stress while adapting to EVs emitted from various cancers under various treatments. Immunizing mice with EVs derived from oxaliplatin-treated B16-OVA cells, for instance, expands IFN-&#947;-producing CD8+ T cells in the spleen, which are further stimulated by ex vivo incubation with ovalbumin, indicating a tumor-specific immune response. Thus, detection of immunogenic EVs b...

The immune system plays a pivotal role in the fight against cancer, both when incited by immune checkpoint inhibition and for the efficacy of conventional cancer therapies. Tumor cells succumbing to genotoxic therapies such as the chemotherapeutic agents oxaliplatin and doxorubicin, or ionizing radiation treatment can release antigens and danger-associated molecular patterns (DAMPs) that potentially initiate an adaptive anti-tumor immune response1. The most prominent DAMPs, in the context of immunogenic cell death, include find-me signals such as chemotactic ATP, eat-me signals such as the exposure of calreticulin, that promotes tumor cell uptake by antigen-presenting cells, and the release of HMGB1, that activates pattern recognition receptors, thereby enhancing the cross-presentation of tumor antigens2. Furthermore, type I interferons (IFN-I), induced via tumor-derived immunogenic nucleic acids or other stimuli, are sensed by dendritic cells, enabling them to effectively prime tumor-specific cytotoxic T cells3,4. Clinically, activated and proliferating CD8+ T cells infiltrating the tumor provide an independent prognostic factor for prolonged survival in many cancer patients. Released from such activated T cells, IFN-&#947; mediates direct antiproliferative effects on cancer cells and drives Th1 polarization and cytotoxic T cell differentiation, thereby contributing to effective immunosurveillance against cancer5,6. Oxaliplatin is a bona fide immunogenic cell death inducer, mediating such adaptive immune response against cancer7. However, the plethora of initial immunogenic signals released by tumor cells under therapy-induced stress remains to be fully unveiled. Despite significant advances in cancer immunotherapy, expanding its benefits to a larger portion of patients remains a challenge. A more detailed understanding of immunogenic signals that initiate T cell activation may guide the development of novel therapies.
A heterogeneous group of membrane-enclosed structures, known as extracellular vesicles (EVs), seem to serve as intercellular communication devices. Emitted by virtually all cell types, EVs carry functional proteins, RNA, DNA, and other molecules to a recipient cell or may alter a cell&#39;s functional state just by binding to receptors on the cell surface. Their biologically active cargo varies significantly by the type and functional state of the generating cell8. In cancer immunology, EVs released from tumor cells have been predominantly regarded as adversarial to immunotherapy because they eventually promote invasive growth, preform metastatic niches9, and suppress the immune response10. In contrast, some studies have shown that EVs can transfer tumor antigens to dendritic cells for effective cross-presentation11,12. EVs may provide immunostimulatory nucleic acids if they emerge under therapy-induced stress, facilitating an anti-tumor immune response13,14. Sensing of such RNA and DNA&#160;innate immune ligands in the tumor microenvironment has recently been shown to modulate responsiveness to checkpoint blockade significantly15,16,17. Hence, the immunogenic role of EVs released by tumor cells under different therapy-induced stress needs to be further elucidated. Since EVs constitute a young yet growing field of research, standardization of methods is still ongoing. Therefore, sharing knowledge is essential to improve the research reproducibility on interactions between EVs and cancer immunology. With this in mind, this manuscript describes a simple protocol to assess the immunogenic effect of tumor-derived EVs in vivo.
This assessment is performed by generating tumor-derived EVs, immunizing recipient mice with those EVs, and analyzing splenic T cells via flow cytometry. EV generation is ideally performed by seeding murine tumor cells in an EV-free cell culture medium for a high degree of purity. Cells are treated with a specific cell stress stimulus, such as chemotherapy, to compare the effect of therapy-induced EVs against baseline immunogenicity of respective tumor-derived EVs. The isolation of EVs may well be performed by various techniques that should be selected according to in vivo applicability and local availability. The following protocol describes a precipitation-based assay with a commercial kit for EV purification. Mice are immunized twice with those EVs. Fourteen days after the first injection, T cells are extracted from the spleen and analyzed for IFN-&#947; production via flow cytometry to evaluate a systemic immune response. With this, the potential of tumor-derived EVs, emerging under different therapeutic regimens, to induce anti-tumor T cell responses is assessed relatively easily, quickly, and with high validity13. Therefore, this method is suitable for an immunological screening of EVs derived from cancer cells under various conditions.

<table><tbody><tr><td>Anti-CD3 FITC</td><td>Biolegend</td><td>100204</td><td>Clone 17A2</td></tr><tr><td>Anti-CD4 PacBlue</td><td>Biolegend</td><td>100428</td><td>Clone GK1.5</td></tr><tr><td>Anti-CD8 APC</td><td>Biolegend</td><td>100712</td><td>Clone 53-6.7</td></tr><tr><td>Anti-IFN&amp;gamma; PE</td><td>eBioscience</td><td>RM90022</td><td>Clone XMG1.2</td></tr><tr><td>Brefeldin A</td><td>Biolegend</td><td>420601</td><td>Brefeldin A Solution (1,000x)</td></tr><tr><td>Cell Strainer, 100 &amp;micro;m</td><td>Greiner</td><td>542000</td><td>EASYstrainer 100 &amp;micro;m</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td>Dulbecco&#039;s Modified Eagle&#039;s Medium with D-glucose (4.5 g/L) and L-glutamine (4 mM)</td></tr><tr><td>FBS Good Forte</td><td>PAN BIOTECH</td><td>P40-47500</td><td>Fetal Calf Serum (FCS)</td></tr><tr><td>Fixable Viability Dye eFluor 506</td><td>eBioscience, division of Thermo Fischer Scientific</td><td>65-0866-14</td><td></td></tr><tr><td>Fixation/Permeabilization Concentrate</td><td>eBioscience</td><td>00-5123-43</td><td>Fixation/Permeabilization Concentrate (10x)</td></tr><tr><td>Fixation/Permeabilization Diluent</td><td>eBioscience</td><td>00-5223-56</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>407952</td><td>From Streptomyces conglobatus - CAS 56092-82-1, &amp;ge; 97% (HPLC)</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-032</td><td>L-Glutamine (200 mM)</td></tr><tr><td>Ovalbumin</td><td>InvivoGen</td><td>vac-pova</td><td>Ovalbumine with &amp;lt; 1 EU/mg endotoxin - CAS 9006-59-1</td></tr><tr><td>Oxaliplatin</td><td>Pharmacy of MRI hospital</td><td></td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>Phosphate Buffered Saline without calcium chloride and magnesium chloride</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>1514-122</td><td>Mixture of penicillin (10,000 U/mL) and streptomycin (10,000 ug/mL)</td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P1585</td><td>Phorbol 12-myristate 13-acetate, &amp;ge; 99% (HPLC)</td></tr><tr><td>PVDF filter, 0,22 &amp;micro;m, for syringes</td><td>Merck Millipore</td><td>SLGV033RS</td><td>Millex-GV Filter Unit 0.22 &amp;micro;m Durapore PVDF Membrane</td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>Invitrogen</td><td>00-4333-57</td><td></td></tr><tr><td>RPMI 1640</td><td>Thermo Fischer Scientific</td><td>11875</td><td>Roswell Park Memorial Institute 1640 Medium with D-glucose (2.00 g/L) and L-glutamine (300 mg/L), without HEPES</td></tr><tr><td>Syringe, 26 G</td><td>BD Biosciences</td><td>305501</td><td>1 mL Sub-Q Syringes with needle (0.45 mm x 12.7 mm)</td></tr><tr><td>Total Exosome Isolation Reagent</td><td>Invitrogen</td><td>4478359</td><td>For isolation from cell culture media</td></tr><tr><td>&lt;em&gt;&amp;beta;&lt;/em&gt;-Mercaptoethanol</td><td>Thermo Fischer Scientific</td><td>31350</td><td>&lt;em&gt;&amp;beta;&lt;/em&gt;-Mercaptoethanol (50 mM)</td></tr></tbody></table>

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.

This protocol is intended to facilitate the straightforward and easily reproducible assessment of the immunogenicity of tumor-derived EVs. Hereby, mice are inoculated with EVs derived from in vitro cultures of tumor cells expressing the model antigen chicken ovalbumin (OVA). The subsequent immune response is analyzed in splenic T cells via flow cytometry.
Figure 1 gives an overview of the practical steps of the entire protocol. Since the work foc...

Watch this Scientific Journal Video about In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells at JoVE.com

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.

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 ...

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JoVE Journal

Cancer Research

2.5K Views.  Technical University of Munich. Deze video laat zien hoe de in vivo immunogeniciteit van tumor-afgeleide extracellulaire blaasjes, of EV's, kan worden beoordeeld met behulp van flowcytometrie. De hierin beschreven screeningsmethode is relatief eenvoudig uit te voeren en kan worden aangepast aan verschillende instellingen. Bij kanker geeft het identificeren van aandoeningen van immunogene extracellulaire blaasjes vrij van bijzonder translationeel belang.Als zodanig kunnen autologe EV's worden gebruikt als gepersonalis...

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Immunology and Infection

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 ...

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the ...

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Adoptive Therapy Using Tumor-Infiltrating Lymphocytes in Pancreatic Cancer Models

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

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

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