This work was supported by NIH R01 CA104757 Grant (M. H. Manjili). We gratefully acknowledge the support of VCU Massey Cancer Center and the Commonwealth Foundation for Cancer Research.

1. Isolation of Lymphocytes5
<ol>
<li>Isolate tumor-draining lymph nodes or spleens from tumor-bearing FVBN202 transgenic mice and prepare single cell suspension in ice-cold RPMI1640 supplemented with 10% FBS. B/I activation in 50-ml polypropylene conical tubes results in a greater T cell yield compared to polystyrene tubes. Ketamine and Xylazine are injected i.p. for anesthesia. Cervical dislocation is used as a method of euthanasia.</li>
<li>Culture the cells (106 cells/ mL) in complete medi...

<ol>
	<li>Goodell, V., Waisman, J., Salazar, L. G., de la Rosa, C., Link, J., Coveler, A. L., Childs, J. S., Fintak, P. A., Higgins, D. M., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Level+of+HER-2/neu+protein+expression+in+breast+cancer+may+affect+the+development+of+endogenous+HER-2/neu-specific+immunity.">Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity.</a> Mol Cancer Ther. 7, 449-454 (2008).</li><li>Disis, M. L., Knutson, K. L., Schiffman, K., Rinn, K., McNeel, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existent+immunity+to+the+HER-2/neu+oncogenic+protein+in+patients+with+HER-2/neu+overexpressing+breast+and+ovarian+cancer.">Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer.</a> Breast Cancer Res Treat. 62, 245-252 (2000).</li><li>Liu, S., Riley, J., Rosenberg, S., Parkhurst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+common+gamma-chain+cytokines,+interleukin-2,+interleukin-7,+and+interleukin-15+for+the+in+vitro+generation+of+human+tumor-reactive+T+lymphocytes+for+adoptive+cell+transfer+therapy.">Comparison of common gamma-chain cytokines, interleukin-2, interleukin-7, and interleukin-15 for the in vitro generation of human tumor-reactive T lymphocytes for adoptive cell transfer therapy.</a> J. Immunother. 29, 284-293 (2006).</li><li>Bear, H. D., Roberts, J., Cornell, D., Tombes, M. B., Kyle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+of+cancer+with+pharmacologically+activated+lymph+node+lymphocytes:+a+pilot+clinical+trial.">Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial.</a> Cancer Immunol Immunother. 5, 269-274 (2001).</li><li>Morales, J. K., Kmieciak, M., Graham, L., Feldmesser, M., Bear, H. D., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+HER2/neu-specific+T+cells+expanded+with+alternating+gamma+chain+cytokines+mediate+tumor+regression+when+combined+with+the+depletion+of+myeloid-derived+suppressor+cells.">Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells.</a> Cancer Immunol Immunother. 58, 941-953 (2009).</li><li>Cha, E., Graham, L., Manjili, M. H., Bear, H. D., Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Cardiff, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+++IL-15+are+superior+to+IL-2+for+the+ex+vivo+expansion+of+4T1+mammary+carcinoma-specific+T+cells+with+greater+efficacy+against+tumors+in+vivo.">IL-7 + IL-15 are superior to IL-2 for the ex vivo expansion of 4T1 mammary carcinoma-specific T cells with greater efficacy against tumors in vivo.</a> Breast Cancer Res Treat. 89, 10578-10582 (2009).</li><li>Kmieciak, M., Morales, J. K., Morales, J., Bolesta, E., Grimes, M., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danger+signals+and+nonself+entity+of+tumor+antigen+are+both+required+for+eliciting+effective+immune+responses+against+HER-2/neu+positive+mammary+carcinoma:+implications+for+vaccine+design.">Danger signals and nonself entity of tumor antigen are both required for eliciting effective immune responses against HER-2/neu positive mammary carcinoma: implications for vaccine design.</a> Cancer Immunol Immunother. 57, 1391-1398 (2008).</li><li>Stern, J. B., Smith, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-2+induction+of+T-cell+G1+progression+and+c-myb+expression.">Interleukin-2 induction of T-cell G1 progression and c-myb expression.</a> Science. 233, 203-206 (1986).</li><li>Kittipatarin, C., Khaled, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+memory+CD8+T+cells+from+lymph+nodes+or+spleen+through+in+vitro+culture+with+interleukin-7.">ex vivo expansion of memory CD8 T cells from lymph nodes or spleen through in vitro culture with interleukin-7.</a> J Immunol Methods. 344, 45-57 (2009).</li><li>Kokaji, A. I., Hockley, D. L., Kane, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+transpresentation+augments+CD8++T+cell+activation+and+is+required+for+optimal+recall+responses+by+central+memory+CD8++T+cells.">IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells.</a> J Immunol. 180, 4391-4401 (2008).</li><li>Le, H. K., Graham, L., Miller, C. H., Kmieciak, M., Manjili, M. H., Bear, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+antigen-sensitized+T+lymphocytes+activated+with+bryostatin+1+++ionomycin+in+IL-7+++IL-15+increases+yield+of+cells+capable+of+inducing+regression+of+melanoma+metastases+compared+to+culture+in+IL-2.">Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2.</a> Cancer Immunol Immunother. 58, 1565-1576 (2009).</li></ol>

Selective expansion of tumor-reactive T cells with effector anti- tumor function can be achieved by the proposed protocol using B/I activation and ex vivo expansion with the gamma chain cytokines IL-2, IL-7 and IL-15. While IL-2 is a T cell growth factor that can support the differentiation and expansion of antigen-specific T cells, IL-7 can inhibit apoptosis of T cells and support their viability during expansion. IL-15 can support memory T cells that are important for generating long-term anti-tumor responses ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bryostatin 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B7431-10ug</td>
<td>&nbsp;</td>
<tr>
<td>Ionomycin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>407950</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-7</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>217-17</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-15</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>210-15</td>
<td>&nbsp;</td>
<tr>
<td>Human IL-2</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>200-02</td>
<td>&nbsp;</td>
<tr>
<td>RPMI1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Gemini</td>
<td>100-106</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>L- glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>&beta;- mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>anti-CD16/32 antibody</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>101302</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-FITC Apoptosis Detection Kit</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>556547</td>
<td>&nbsp;</td>
<tr>
<td>FITC-CD4</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100406</td>
<td>&nbsp;</td>
<tr>
<td>PE-CD8</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100708</td>
<td>&nbsp;</td>
<tr>
<td>anti-c-Erb2/c&#x2013;Neu</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>OP16</td>
<td>&nbsp;</td>
<tr>
<td>PE- anti mouse IgG</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>405307</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>04018</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Hycor</td>
<td>87144</td>
<td>&nbsp;</td>
<tr>
<td>Light microscope</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>V200073</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IFN-&gamma; ELISA set</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>555138</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture flasks</td>
<td>&nbsp;</td>
<td>Greiner</td>
<td>658175</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Watch this Scientific Journal Video about Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation at JoVE.com

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

ex-vivo-expansion-tumor-reactive-t-cells-means-bryostatin-1ionomycin

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Immunology and Infection

14.6K Views.  Virginia Commonwealth University- Massey Cancer Center. An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Video: Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast...

ex vivo t cell stimulation in the pancreatic tumor: a method to increase cytokine production in t cells

This article describes the technique for ex vivo T cell stimulation to increase the intracellular concentration of cytokines such as interferon-gamma or IFN-gamma and tumor necrosis factor-alpha or TNF-alpha in the T cell. These cytokines&#160; mediate an effective anti-tumor response and can have a therapeutic role in the...

Ex Vivo T Cell Stimulation in the Pancreatic Tumor: A Method to Increase Cytokine Production in T Cells

expansion and enrichment of gamma-delta (&#947;&#948;) t cells from apheresed human product

Expansion and Enrichment of Gamma-Delta &amp;#947;&amp;#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

generation of orthotopic pancreatic tumors and ex vivo characterization of tumor-infiltrating t cell cytotoxicity

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...

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

assessing the innate sensing of hiv-1 infected cd4+ t cells by plasmacytoid dendritic cells using an ex vivo co-culture system.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

in vivo immunogenicity screening of tumor-derived extracellular vesicles by flow cytometry of splenic t cells

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...

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

an ex vivo technique to generate tumor-specific chimeric antigen receptor t cells

This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host...

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

ex vivo expansion of natural killer cells from peripheral blood mononuclear cells

This video demonstrates a technique for the expansion of natural killer (NK) cells directly from peripheral blood mononuclear cells (PBMCs) using a feeder cell line. PBMCs are co-cultured with genetically modified lymphoblastoid feeder cells expressing membrane-bound interleukin-21 (mIL-21), and the culture is supplemented with the cytokines interleukin-2 (IL-2) and interleukin-15 (IL-15). Together, this cocktail of ligands leads to the prolonged expansion of NK cells with high...

Ex Vivo Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells

Incubate at room temperature for 30 minutes, then, remove the BSA by firmly upending the plate before adding 2 milliliters of PBS to wash. After collecting the viral supernatant from the HEK293T cells, add fresh TCM to a final volume in milliliters equal to the number of RHFF-coated wells plus an additional 3...

purification and expansion of mouse invariant natural killer t cells for in vitro and in vivo studies

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo...

isolation and flow cytometric analysis of human endocervical gamma delta t cells

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell...

After centrifugation, save 1 x 106 PBMCs for flow cytometry, and mix 5 x 106 PBMCs with 10 x 106 100 gamma-irradiated 221-mIL-21 cells in a special 6-well plate. Add 30 milliliters of R-10 media supplemented with human interleukin-2 and human interleukin-15 to the same 6-well plate, and incubate the plate at 37 degrees Celsius with 5% carbon dioxide with replacing the media every three to four...

isolation and culture expansion of tumor-specific endothelial cells

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

ex vivo expansion and genetic manipulation of mouse hematopoietic stem cells in polyvinyl alcohol-based cultures

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and...

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

ex vivo expansion of hematopoietic stem cells from human umbilical cord blood-derived cd34+ cells using valproic acid

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

defining gene functions in tumorigenesis by ex vivo ablation of floxed alleles in malignant peripheral nerve sheath tumor cells

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

isolation and ex vivo culture of v&#948;1+cd4+&#947;&#948; t cells, an extrathymic &#945;&#946;t-cell progenitor

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture...

artificial antigen presenting cell (aapc) mediated activation and expansion of natural killer t cells

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

an in vitro assay to evaluate the anti-tumor activity of tumor-specific car t cells

This video demonstrates an in vitro assay to validate chimeric antigen receptor (CAR) T cell activity. B-cell lymphoma cells expressing CD19 antigen and cytoplasmic luciferase are co-cultured with CD19-specific CAR T cells. CAR binding to CD19 on a tumor induces the CAR T cells to release cytokines and toxins that cause tumor cell cytotoxicity. The CAR T cell activity is evaluated by quantifying the released cytokine and decreased luciferase-mediated bioluminescence in the tumor...

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.