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&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;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>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</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&amp;reg; 50 &amp;mu;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. Wij presenteren hier een aanvraag voor een standaard immunologische techniek (CFSE gekleurd OT-ik proliferatie) willen snel controleren adjuvans-gemedieerde cytotoxische T lymfocyten (CTL) generatie in vivo. Deze snelle inschatting van CTL capaciteiten is nuttig voor de ontwikkeling van profylactische vaccins tegen intracellulaire pathogenen, alsmede therapeutische kanker vaccins.

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

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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

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

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

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

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

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

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

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

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

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

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

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

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

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

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

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

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

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