We are indebted to our technical assistants:&#160;U. Br&#246;der and H. Shkarlet, who helped us during experimental procedures. This work was partly funded by EU grants (UniVax, contract No. 601738, and TRANSVAC2, contract No. 730964), and a Helmholtz Association grant (HAI-IDR). The funding sources did not influence the research design, generation of the manuscript or decision to submit it for publication.

All mice used in this study were from the C57BL/6 background. All the animals were kept under pathogen-free conditions. All experiments were performed according to the normative of the German animal protection law (TierSchG BGBl. I S 1105; 25.05.1998) and were approved by the Lower Saxony Committee on the Ethics of Animal Experiments and the state office (Lower Saxony State Office of Consumer Protection and Food Safety), under permit number 33.4-42502-04-13/1281 and 162280.
1. CFSE Staining of OT-I T Cells and Adoptive Transfer
NOTE: OT-I mice are transgenically generated animals that express a T cell receptor (TCR) with fixed &#945; and &#946; chains that together recognize the immuno-dominant peptide of OVA, SIINFEKL10,11. As a result, these mice have a considerably high number of SIINFEKL-specific CD8+ T cells (97%)12 when compared to normal or OVA-vaccinated mice (&#8804; 1%)13.
<ol>
	<li>Isolation of traceable CD8+ T cells from OT-I mice expressing T lymphocyte-specific Thy1a (Thy1.1) allele:
	<ol>
		<li>Euthanize 6-9 weeks old OT-I mice by CO2 inhalation followed by cervical dislocation14. Dissect the spleen and major lymph nodes (inguinal, axillary and cervical pairs only) by cutting the skin with surgical scissors, detaching the skin from the body with the help of surgical pliers and forceps and place them in Petri dishes (60 x 15 mm) each containing a 100 &#181;m pore mesh cup for tissue grinding and cell release.</li>
		<li>Maintain petri dishes (each with one mesh cup) in 3-5 mL of complete RPMI medium (RPMI 1640, 10% v/v FCS, 100 U/mL penicillin, 50 &#181;g/mL streptomycin) kept on ice.</li>
		<li>Mash the organs with the help of a syringe plunger or a similar sterile instrument (prior cutting is not needed) and collect the resulting single cell suspension in 15 mL centrifuge tubes.</li>
		<li>Centrifuge the cell suspension at 300 x g for 10 min at 4 &#176;C. Discard the supernatant. Wash the cells in 10 mL of cold PBS by centrifugation and lyse the erythrocytes from the spleen by re-suspending the pellet in 1 mL/spleen of ammonium chloride buffer (ACK buffer, commercially available). Incubate the cells for 1.5 min on ice and subsequently wash the cells with 10 mL of cold PBS by centrifugation (as done before).</li>
		<li>Re-suspend the pellet in the same tube in 1 mL of PBS (pH 7.2) containing 5% fetal bovine serum for magnetic isolation.</li>
	</ol>
	</li>
	<li>To perform magnetic isolation, proceed to the negative selection of CD8+ T cells by using a magnetic isolation kit according to the manufacturer instructions or published protocols15.</li>
	<li>To perform CFSE staining, first count the number of CD8+ T cells obtained from OT-I mice by using an automated cell counter (particle counter). Stain 1 - 5 x 107 cells/mL in a volume of 1-5 mL with 5 &#181;M CFSE in PBS for 7 min at 37 &#176;C, protected from light in a 15-ml falcon tube.</li>
	<li>Quench the CFSE staining by adding the same volume (1:1) of fetal bovine serum to the cells, and incubate them for additional 7 min at 37 &#176;C protected from light. Wash the cells with 10 mL of PBS twice.</li>
	<li>Count the cells using an automated cell counter. Set the cell number to 3-5 x 107 cells/mL of PBS in order to inject 3-5 x 106 cells/mouse in 100 &#181;L intravenously (i.v.) via the tail vein.</li>
	<li>To perform tail vein injections, immobilize the mice in appropriate restrainers. Warm the back area and tail of the mice to be injected by using a red-light lamp, placed between 20 to 25 cm away from the mice for 1-3 min to allow tail vein vasodilation. 
	NOTE: This helps to ease vein detection and administration of the cell suspension.</li>
	<li>Ensure (with the hand placed in between the mice and the lamp) that it is not too hot for the mice and when the tail veins are clearly visible, proceed to injection. Inject the cells into the lateral or dorsal tail vein using an 1mL syringe with a 25-gauge needle16.</li>
</ol>
2. Immunization (Endo-free OVA +/- Adjuvant)
<ol>
	<li>Place the mice transplanted with OT-I cells (step 1.7, Figure 1) in an anesthesia chamber and administer isoflurane in oxygen by an anesthesia machine14.</li>
	<li>When the mouse is completely asleep under anesthesia, take it out from the chamber and shave the fur of the mouse on the area over the gluteus superficialis (lateral lower back) by using an electric hair trimming machine, in order to perform a clean injection with a good view of the application area.</li>
	<li>Inject 50 &#181;L of the vaccine, s.c. in the shaved area using a 25-gauge needle.</li>
</ol>
3. Isolation of Lymphocytes and Staining for Flow Cytometry Analysis
<ol>
	<li>Isolation of lymphocytes and splenocytes
	<ol>
		<li>Euthanize the vaccinated mice by CO2 inhalation followed by cervical dislocation.</li>
		<li>Extract the draining and distant lymph nodes and spleen and place them in separate Petri dishes containing 100 &#181;m pore mesh cups for tissue grinding and cell release. Follow steps 1.1.2 through 1.1.3.</li>
		<li>Decant the supernatant after centrifugation and re-suspend the cell pellets in the same volume of remnant PBS (approx. 100 &#181;L).</li>
	</ol>
	</li>
	<li>Staining for flow cytometry analysis
	<ol>
		<li>In a 15-mL centrifuge tube prepare a master mix containing the staining antibodies in the concentrations depicted in Table 1, thus yielding a 2x concentrated staining mix. Prepare enough volume of master mix to have 100 &#181;L per sample. Mix the cells and the master mix 1:1 and incubate it at 4&#176;C for 30 min. 
		NOTE: The final staining volume per sample should be 200 &#181;L (100 &#181;L of cell pellet +100 &#181;L of antibody master mix).</li>
		<li>Wash the cells twice by centrifugation as described in 1.1.3, adding 10 mL of PBS, twice.</li>
		<li>Re-suspend the stained cells in 0.5-1 mL of PBS for acquisition and transfer the suspension to flow cytometer tubes. Always keep the samples on ice and protected from light.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry
<ol>
	<li>Always pre-filter the cell samples using 70-100 &#181;m filters.</li>
	<li>Prepare single staining compensation controls for the fluorophores detailed in Table 1 (beads or cells). 
	NOTE: Compensation should be performed in cytometer or analysis software17,18,19 and applied to all samples.</li>
	<li>Follow the gating strategy depicted in Figure 2. Briefly, gate populations in forward scatter height vs. forward scatter area (Figure 2A), and again side scatter wide vs. side scatter area (SSA, Figure 2B) in order to exclude doublets.</li>
	<li>Gate the population from Figure 2B by plotting BV 650 (auto-fluorescence) vs. FITC (CFSE) in order to discriminate true CFSE stained cells from high auto-fluorescent cells. Include beads or cells stained with antibody conjugated to BV 650 into compensation controls. This plot (Figure 2C) of BV 650 vs. FITC is used to gate the OT-I CFSE stained cells.</li>
	<li>Gate the population from Figure 2C for Pe-Cy7 (Thy1.1 -CD90.1-) vs. APC (CD8) in order to distinguish cells derived from donor vs. recipient mice. 
	NOTE: Cells derived from OT-I donor mice are Thy1.1+ (CD90.1), whereas WT (C57BL/6, recipient) mice-derived cells are Thy1.2+ (CD90.2) (Figure 2D). Some laboratories have OT-I mice in a Thy1.2 background and WT (C57BL/6) in a Thy1.1. In this case you have to use an antibody against Thy1.2 for OT-I cell gating.</li>
	<li>Re-gate CD8+ cells from Thy1.1+ population by plotting it on a gate comprising BV 450 (CD4) vs. APC (CD8) (Figure 2E).</li>
	<li>Display the population gated in Figure 2E by using a histogram display of the CD8+ cells showing CFSE (FITC, 530/15 channel -blue laser-), gate the proliferated population by including intensities from 102 up to the level where the undivided control populations are (intensities ~105, depending on the efficacy of the CFSE staining and the voltage setting on the flow cytometer) (Figure 2F).</li>
	<li>Make an additional gate (not displayed in figure 2) plotting FITC vs. L/D marker (450/20 channel, UV laser) in order to assess dead cells in parallel to the proliferation evaluation.</li>
	<li>Acquire at least 5000 events for the compensation controls and 10.000 events (gate E, Figure 2) from the samples at a flow cytometer. Run the cytometer at low or medium flow (do not exceed flow rates of 20.000 events/s).</li>
</ol>

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The authors have nothing to disclose.

Modern vaccines are ideally composedof purified antigen and adjuvants, with the possible addition of a delivery system like liposomes, virus-like particles, nanoparticles or live vectors. A key aspect when designing a vaccine is to choose the right adjuvant according to the clinical needs. Part of the scope could involve favoring a humoral vs. cellular immune response (or both), the election of a local vs. a systemic immune response (or both), and the kind of memory that the vaccine must generate in the target population. One crucial aspect of adjuvant evaluation is to rapidly determine its capacity to generate CTL. We presented here a method based on already known techniques, to rapidly determine the features of the CTL response in vivo in a mouse model by measuring OT-I CD8 T cell proliferation. This method allows for the prediction of the potency of the immune response elicited by adjuvants (proliferation of OT-I T cells) in only 4 working days. This method further facilitates the comparison of the actions of adjuvants in terms of the immune response (local vs. systemic) and its effective action. Here, we have showed an example on how immunization with different adjuvant treatments (ADJ1 vs. ADJ2) will affect the immune response. The action of ADJ2 was restricted to the local area of administration where it activates the immune response in the draining lymph nodes, whereas the action of ADJ1 with the same dose was more widespread, generating a CTL response both at the local and systemic level but with less potency than ADJ2 in the draining lymph nodes.
Critical steps for the successful evaluation of an adjuvant&#39;s CTL capacity are the choice of young OT-I animals (as a source of CD8 T cells) and the use of endotoxin-free OVA for immunization. Since OT-I mice are transgenic animals generated to produce CD8 T cells that recognize OVA peptide SIINFEKL in a MHC-I context, its artificial selection of the mouse TCR increases the appearances of hyper proliferative CD8+ T cells20 with age. An inflammation of the axillary lymph nodes is thus a more common feature of aged OT-I mice. Taking this into account, it is recommended to use young OT-I animals when possible (6-9-week-old animals) and to avoid isolating cells from any enlarged organs (either spleen of lymph nodes). The use of enlarged organs with proliferating CD8 T cells will affect the whole assay since most likely differences in proliferation between controls and treatments will not be obtained. A similar pitfall for the discrimination of adjuvant CTL generation capacity could be generated by using OVA protein with traces of endotoxin, which can elicit a more potent immune response as compared to endotoxin-free OVA21,22 (see Materials and Reagents), since endotoxins are strong immuno-modulators per se 23. Therefore, the OVA protein used for immunization must be endotoxin-free. The use of 20 or 50 &#181;g of endotoxin-free OVA depends on the immunization route being 50 &#181;g a suitable dose for subcutaneous testing of adjuvants. Additionally, the use of high concentrations of CFSE has been reported in the literature to kill the stained cells24 and could also result in the failure of the assay.
Some modifications or improvements to different techniques used in the protocol were implemented in order to reduce the stress of the animals, to increase reproducibility of the experiments. For example, to minimize the heating of animals by just heating the back and the tail of the mice and not the whole animal, or to avoid subcutaneous injection related stress by anesthetizing the animals before vaccination. The anesthesia is not required by animal welfare regulations for a subcutaneous injection but in our experience, it improves reproducibility by decreasing variations introduced by the stress response.
A great limitation of this method is that since CFSE stains the membrane of cells, its high brightness usually impairs the detection of signals from the cytosol. Therefore, if the confirmation of CTL capacity is needed, the secretion of IFN-&#947; should be evaluated by a specific secretion assay 25, and not by intracellular cytokine staining.
The use of the measurement of proliferation to estimate the CTL generation capacity by adjuvants in just 4 days has the advantage of shorter time needed than traditional CTL assays26 and it is a good prospective experiment in the case where further confirmation by other methods is needed.
Although restricted to OVA as an antigen, and therefore to the use of OT-I T cells, the method presented here allows the study of the properties of the activation induced by different adjuvants after sorting of the proliferated cells. One of the possible applications is the study of the metabolic signatures induced by different adjuvants in the proliferated OT-I T cells and its relationships with the development of memory27. Additional secondary screenings could evaluate the biological properties of the stimulated cells, such as the expression of cytokines or degranulation markers by flow cytometry or their cytotoxic capacity by performing in vivo CTL tests8,26,28.Since this application has the limitation of its use in mice, extrapolations to humans can be performed by exploiting screenings based on humanized mice29,30,31.
This method as we have presented here is robust enough to be used as a first screening for the selection of cellular response activators and also flexible enough to be modified or coupled to further analysis of the proliferated T cells. In summary, this rapid in vivo assessment of adjuvant CTL generation capacity is an ideal tool for vaccine laboratories in academia and industry that are interested in a fast characterization of the mode of action of their adjuvant candidate molecules.

Cytotoxic T lymphocytes (CTL) inducing vaccines are key therapeutic interventions that have been developed to fight certain types of cancer1. CTL are also important for prophylactic vaccines against intracellular pathogens2. Moreover, CTL are one of the few immune defense mechanisms functionally active in risk populations such as neonates3,4 whom also depend on CTL to combat early life infections5. In this regard, vaccines against Respiratory Syncytial Virus (RSV) that were developed with an adjuvant that does not elicit CTL responses (alum) resulted in a complete failure of the vaccine leading to serious complications upon infections in infants6. These negative effects of vaccination can be reversed by a CD8+ T cell response7. We have previously demonstrated that the main cytokines (type I interferons) elicited by some stimulator of interferon genes (STING) agonists are essential for the CTL responses generated by these adjuvants8, in part by measuring the proliferation of OT-I T cells after vaccination and using these results as a measure of CTL inducing capabilities observed in extended vaccination schedules9. The measurement of the proliferation of OT-I CD8+ T cells in a wild type (WT) C57BL/6 recipient mouse by carboxyfluorescein succinimidyl ester (CFSE) dye dilution is a robust estimation of the capability of the adjuvant of a vaccine to generate cross-priming of SIINFEKL, (the immuno-dominant peptide of ovalbumin, OVA). Variations of this technique are widely used for the assessment of the proliferation of OT-I CD8+ and OT-II CD4+ T cells. For example, it has been used in the absence of selected cytokines (KO mice) or to measure vaccine efficacy after antigen recall in WT animals. We devised a short protocol (4 days experiment) in which after passive transfer of CFSE-stained OT-I CD8+ T cells, a subcutaneous (s.c.) immunization consisting of one dose of 50 &#181;g of endotoxin-free OVA supplemented with test adjuvants is administered (Figure 1). The follow up of the results 48 h after vaccination provides a reliable proof of the capacity of the adjuvant to generate CTL responses. By this strategy, it is possible to assess the potency of the local immune response in the draining lymph node after immunization as well as the extent of the response by measuring the CTL activity in the spleen (or distant lymph nodes).

<table>
	<tbody>
		<tr>
			<td>BD LSR Fortessa Cell Analyzer</td>
			<td>BD</td>
			<td>Special Order</td>
			<td>Flow Cytometer</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes</td>
			<td>C34554</td>
			<td>Proliferation Dye</td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Molecular Probes</td>
			<td>L23105</td>
			<td>Dead Cell Marker</td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0900-82</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a Antibody</td>
			<td>BioLegend</td>
			<td>100712</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>BV421 Rat Anti-Mouse CD4</td>
			<td>BD</td>
			<td>740007</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Z2 coulter Particle count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>9914591DA</td>
			<td>Cell counter. Z2 Automated particle/cell counter</td>
		</tr>
		<tr>
			<td>EndoGrade Ovalbumin (10 mg)</td>
			<td>Hyglos(Germany)</td>
			<td>321000</td>
			<td>Ovalbumin endotoxin free tested.</td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;m pore mesh cups).</td>
		</tr>
		<tr>
			<td>Sample Vials</td>
			<td>Beckman Coulter</td>
			<td>899366014</td>
			<td>Sample vials for Z2 automated counter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice (CD90.2)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Company is now Envigo</td>
		</tr>
		<tr>
			<td>OT-I (C57BL/6 background, CD90.1)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Inbreed at our animal facility. Company from where adquired is now Envigo</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fischer (Corning)</td>
			<td>14-959-5</td>
			<td>Corning Falcon Round-Bottom Polystyrene Tubes</td>
		</tr>
		<tr>
			<td>Falcon 15 mL tubes</td>
			<td>Fischer (Corning)</td>
			<td>05-527-90</td>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>PBS (500 mL)</td>
			<td>Fischer (Gibco)</td>
			<td>20-012-027</td>
			<td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td>
		</tr>
		<tr>
			<td>Red lamp (heating lamp)</td>
			<td>Dirk Rossmann GmbH (Germany)</td>
			<td>405096</td>
			<td>Heating infrred lamp (100 wats)</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane)</td>
			<td>Abbott Laboratories (USA)</td>
			<td>5260.04-05.</td>
			<td>Isoflurane anesthesic (250 mL flask).</td>
		</tr>
		<tr>
			<td>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</td>
			<td>Parkland Scientific</td>
			<td>V3000PK</td>
			<td>Isoflurane anesthesia machine.</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>11-875-093</td>
			<td>Base medium with Glutamine (500 mL)</td>
		</tr>
		<tr>
			<td>Pen-Strept antibiotic solution (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>15-140-148</td>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>10082147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer (100 ml)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>A1049201</td>
			<td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td>
		</tr>
		<tr>
			<td>Plastic Petri Dishes</td>
			<td>Nunc (distributed by ThermoFischer)</td>
			<td>150340</td>
			<td>60 x 15mm Plastic Petri Dish, Non-treated</td>
		</tr>
		<tr>
			<td>Cell Clump Filter</td>
			<td>CellTrics (Sysmex)</td>
			<td>04-004-2317</td>
			<td>CellTrics&#174; 50 &#956;m, sterile</td>
		</tr>
	</tbody>
</table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

In order to test the treatments using a different combination of adjuvants (ADJ1 and ADJ2), we have assessed the CTL generation capacity by measuring the proliferation of adoptively transferred OT-I CD8+ T cells by flow cytometry (Figure 2). For this, we previously stained isolated cells from the draining lymph nodes and spleen (Table 1). By measuring the proliferation of CD8+ T cells in lymph nodes and spleen, we were able to corroborate a higher CTL generation capacity of ADJ2 in the draining lymph nodes (Figure 3) when compared to ADJ1, OVA alone or PBS control. In contrast, we have observed that ADJ2 was not able to generate a CTL response in a systemic compartment (spleen), whereas ADJ1 was showing high levels of proliferation by splenic CD8+ T cells, not only compared to the controls (PBS and OVA) but also superior to ADJ1. By dissecting the action of ADJ2 using this rapid in vivo proliferation assay, we can confirm its strong action as a local but not a systemic CTL generator. Moreover, ADJ1 acts both at the local site of injection (draining lymph nodes) as well as systemically with an increased OT-I proliferation in the spleen. The obtained results allow us to characterize the adjuvant&#39;s activity and its extent, exemplified by the observed effects of ADJ2 (local) and ADJ1 (systemic).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57401/57401fig1.jpg" /> 
Figure 1: The assay timeline. The assay timeline represents the initial OT-I T cell transfer at day 0, the s.c. vaccination at day 1, and the sampling spleen and draining lymph nodes 2 days later. <a href="/files/ftp_upload/57401/57401fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57401/57401fig2.jpg" /> 
Figure 2: Flow diagram of the gating strategy followed to measure the proliferation of CD8+ T cells (OT-I, Thy1.1+) by flow cytometry. Two samples are represented in different colors (red and light blue) for a better visualization. A-B. Single cells are discriminated from doublets successively in the first two gates by plotting forward-scatter-height vs. forward-scatter-area and side-scatter-width vs. side-scatter-area. C. Cells gated in B are displayed by their fluorescence intensities of BV 650 channel (auto fluorescence) plotted against their CFSE intensity (where diming indicates cells divisions/proliferation) in the 530/30 YG channel. This gate allows the selection of true CFSE positive cells by discriminating those that have high auto fluorescence. D. CFSE positive cells were gated with their high fluorescence intensity of Pe-Cy7 (Thy1.1, marker for OT-I cells) vs. their APC intensity (CD8). E. BV 450 (CD4) plotted against APC (CD8) for previously gated Thy1.1 positive cells to accurately select CD8+ T cells. F. Previously gated CD8+ cells are plotted in a histogram against the CFSE intensity to finally gate the proliferated population. <a href="/files/ftp_upload/57401/57401fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57401/57401fig3.jpg" /> 
Figure 3: Adjuvant driven CTL generation. The ability to elicit local or systemic CTL responses is depicted for 2 different adjuvant treatments (ADJ1 and ADJ2) along antigen and PBS controls. The proliferation of the adoptively transferred OT-I T cells is examined in the draining lymph node (inguinal, for the exemplified subcutaneous (s.c.) administration) and in the spleen, as indicated in the figure rows. The cell proliferation is measured in all relevant treatments (columns) in order to accurately compare the extent of the immune response in its local (draining lymph node) or systemic (spleen) range of action. <a href="/files/ftp_upload/57401/57401fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dyes - Antibodies</td>
			<td>Clone</td>
			<td>Fluorophore/channel-filter</td>
			<td>Concentration 2X</td>
		</tr>
		<tr>
			<td>Thy1.1 (CD90.1)</td>
			<td>HIS51</td>
			<td>PE-Cy7 - 780/60 YG</td>
			<td>1:750</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>53-6.7</td>
			<td>APC - 670/14 R</td>
			<td>1:280</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>RM4-5</td>
			<td>BV 421 - 450/50 V</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>-</td>
			<td>FITC - 530/30 YG</td>
			<td>(according to CFSE-staining protocol)</td>
		</tr>
		<tr>
			<td>DCM (Dead Cell Marker)</td>
			<td>-</td>
			<td>-/ U.V. - 450/50 UV</td>
			<td>1:500</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody stainings for flow cytometry. Fluorophore-conjugated antibody clones used and recommended staining concentrations (2x).

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

Nanyang Technological University

Research

JoVE Journal

Medicine

8.6K Views.  Helmholtz Centre for Infection Research. We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Video: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...