We thank Lucile Wrenshall for the use of the plate reader, Nancy Bigley for useful discussion, and Tom Brown for the gift of ICR mice. This work was funded by grant 1R01AI114804 from the National Institute of Allergy and Infectious Diseases and by WSU Faculty Development funds (to J.A.K.). J.N.G. was supported in part by the National Institute of General Medical Sciences grant R25GM090122.

All experiments are carried out in accordance with protocols approved by the Wright State University Lab Animal Care and Use Committee and Institutional Review Board.

NOTE: Human PBMCs are isolated by the Ficoll density gradient centrifugation method5.
1. Spleen Harvest
<ol>
	<li>House adult female ICR mice in standard lab conditions with the food, water, and bedding requirements specific to the species. 
	NOTE: Animals had a mean age of 2.9 months and a mean weight of 30.4 g at the time of euthanasia.</li>
	<li>On the date of isolation (DOI), euthanize the animals individually without other conspecific animals present. Make all efforts to ensure minimal stress to the mouse. Euthanize using CO2 with secondary cervical dislocation to ensure death.
	<ol>
		<li>Place the animal, still inside the transfer cage, into the CO2 chamber and start the gas flow at 5 L/min. This flow rate displaces oxygen at the recommended 10-30% per min.</li>
		<li>After the animal is unconscious, increase the CO2 flow rate to 15 L/min. Check the animal for cessation of breathing and leave in the chamber for an additional 2 min.</li>
		<li>Apply cervical dislocation with distraction force to ensure death.</li>
	</ol>
	</li>
	<li>Harvest the spleen from the animal using aseptic technique within 10 min of death.
	<ol>
		<li>Sterilize two sets of scissors and forceps for the removal of the spleen in an autoclave oven prior to use. Sterilize the dissection surface by applying 70% ethanol.</li>
		<li>Apply 70% ethanol to the mouse abdomen. Using one set of scissors and forceps, make a cut in the fur and skin from the left side of the abdomen, then pull apart and peel back the skin to reveal the peritoneum. Once exposed, apply 4% chlorhexidine solution before opening the peritoneum.</li>
		<li>Using the second set of scissors and forceps, make a 2- to 3-cm cut along the central abdomen. Open the peritoneum and remove the spleen by cutting away visceral attachments and excess fat. Place the spleen in Dulbecco&#39;s phosphate-buffered saline (DPBS).</li>
	</ol>
	</li>
</ol>
2. Preparation of Raw Splenocytes
NOTE: Perform all steps under a laminar flow biosafety cabinet using aseptic technique.
<ol>
	<li>Soak the harvested spleens in 10 ml of deionized sterile H2O for 5 min in a 10 cm culture dish to lyse the surface erythrocytes. 
	NOTE: This step can be omitted if the spleen was damaged during isolation.</li>
	<li>To release the splenocytes, crush the spleens between two sterilized glass slides (frosted side facing the spleen) over a fresh 10 cm culture dish. Mix with 10 ml of RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 &#181;g/ml streptomycin (below referred to as complete RPMI).</li>
	<li>Filter the cells through a sterile 40 &#181;m nylon cell strainer into a new 10 cm culture dish to remove the connective tissue and debris.</li>
	<li>Lyse the remaining erythrocytes by adding 20 ml of RBC lysis buffer composed of 155 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA at pH 7.4 and ~300 mOsm/L. 
	NOTE: Osmolality is measured by a freezing point or a vapor pressure osmometer.
	<ol>
		<li>Transfer the cells from the 10-cm plate to a 50 ml conical tube and centrifuge at 250 x g for 10 min (4 &#176;C).</li>
		<li>Remove the supernatant, add 20 ml of RBC lysis buffer, and re-suspend the pelleted cells by pipetting.</li>
		<li>Incubate at room temperature in the lysis buffer for 10 min with gentle rocking.</li>
		<li>Centrifuge for 10 min at 250 x g (4 &#176;C) to pellet the cells. Pour off the lysis buffer.</li>
		<li>Re-suspend cells in 10 ml of complete RPMI and centrifuge again (250 x g, 10 min, 4 &#176;C). Discard the supernatant.</li>
	</ol>
	</li>
	<li>Re-suspend the splenocytes in 2 ml of complete RPMI warmed to 37 &#176;C for transfer to the nylon wool fiber column.</li>
</ol>
3. Purification of T Lymphocytes
<ol>
	<li>Wash the nylon wool columns twice with 5 ml of complete RPMI. Incubate at 37 &#176;C in a 5% CO2 cell culture incubator for 1 hr. 
	NOTE: It is imperative to keep the nylon wool wet for the duration of the experiment and to use complete RPMI warmed to 37 &#176;C for all nylon wool isolation steps.</li>
	<li>Add isolated splenocytes to the column and incubate for 1 hr to allow the B cells, fibroblasts, and accessory cells to adhere to the nylon wool.
	<ol>
		<li>Add 2 ml of RPMI containing the splenocytes into the column and pass through until the top of the nylon wool is reached.</li>
		<li>Add 2 ml of complete RPMI warmed to 37 &#176;C on top of the nylon wool and pass the medium through the wool until the liquid level reaches the top surface.</li>
		<li>Add 3 ml of warm complete RPMI to the column to completely cover the nylon wool.</li>
		<li>Incubate the loaded column undisturbed in a 37 &#176;C, 5% CO2 cell culture incubator for 1 hr.</li>
	</ol>
	</li>
	<li>Elute the cells by washing the column twice with 5 ml of complete RPMI. Perform an additional wash of the eluted cells by centrifugation.
	<ol>
		<li>Fill the column to the top 2 times with complete RPMI and allow the solution in the column to flow through into a 50 ml sterile conical tube. 
		NOTE: Avoid tapping or bumping the column, which can dislodge the B cells, accessory cells, and fibroblasts adhering to the nylon wool.</li>
		<li>Collect the flow-through fraction and spin at 250 x g for 10 min (4 &#176;C) to pellet the cells. Wash once with 10 ml of complete RPMI. Pellet the cells again (250 x g, 10 min, 4 &#176;C) and re-suspend in 2 ml of complete RPMI.</li>
		<li>Measure the cell density using a hemocytometer and dilute in complete RPMI to 0.5 x 106 cells/ml.</li>
		<li>Seed 1 or 2 ml aliquots of cells into each well of a 24- or 6-well cell culture plate, respectively, and culture the cells at 37 &#176;C with 5% CO2. 
		NOTE: 1 mM 1,4-dithiothreitol (DTT) can be added to each well at this stage to improve T-cell survival.</li>
		<li>Optional: Confirm the efficiency of the isolation protocol by flow cytometry. This protocol normally yields ~80% T lymphocytes23. 
		NOTE: Nylon wool-purified cells contain approximately 50% CD4+ and 23% CD8+ cells. To purify the CD4+ and CD8+ subpopulations further, a single immunomagnetic depletion step can be performed using eight antibodies and magnetic beads24. Alternatively, purer CD4+ and CD8+ T-cell populations can be isolated by positive or negative selection with specific antibodies.</li>
	</ol>
	</li>
</ol>
4. Activation of T Lymphocytes and Experimental Drug Exposure
<ol>
	<li>Activate T lymphocytes within 24 hr of isolation through the addition of PMA and ionomycin calcium salt or magnetic beads coated with anti-CD3 and anti-CD28 antibodies23,25,26. Add experimental drugs (e.g., CsA, FK506, rapamycin, TRAM-34, and FTY720) at the time of activation. 
	NOTE: Here, PMA concentrations varied from 2 to 250 ng/ml, and the ionomycin concentration was kept at 250 nM. If possible, dilutions should be prepared from stock aliquots of each drug so that the volume added to each well of cells is &#8805;1 &#181;l to ensure reproducibility. The anti-CD3/anti-CD28-coated magnetic beads are added at a 1:1 bead-to-cell ratio. Increasing the number of beads per cell (i.e., the bead-to-cell ratio) will increase the intensity of stimulation25,26. The beads are washed and re-suspended in complete RPMI before their addition to the cells. Note that trypan blue stains the beads.</li>
	<li>Culture the cells for 12-72 hr at 37 &#176;C with 5% CO2 before analyzing with the automated cell counter.</li>
</ol>
5. Automated Cell Counter Data Collection
<ol>
	<li>Before running the sample, make sure that the cells are gently mixed with a serological pipette to avoid clumps. This is particularly important for activated lymphocytes because of their increased adhesiveness.
	<ol>
		<li>For cultures that are activated with anti-CD3/CD28 beads, after pipetting, transfer the sample to a 1.5 ml or 2 ml centrifuge tube. Separate the beads by keeping the tube on a magnet for 1-2 min. Use the supernatant containing the cells for analysis. 
		NOTE: Analyze both resting and activated cells within 1 hr to account for any pre-activation of the resting cells by serum present in the culture medium. After 12 hr of PMA/ionomycin stimulation, a small increase in cell size was detectable (Figure 2). At 72 hr, no significant cell size increase compared to 48 hr was observed (data not shown).</li>
	</ol>
	</li>
	<li>Transfer 1 ml of the cell suspension into a sample cup and run it through the automated cell counter according to the instructions in the manual. 
	NOTE: For each of the trials, a minimum of 1 ml (maximum of 2 ml) of cell culture should be used. The automated cell counter uses a syringe to mix trypan blue with the cell suspension and passes the cell-trypan blue mixture over a field that is imaged and analyzed by the software. The software detects trypan blue-stained cells, draws a circle around each cell, and determines the diameter. 100 images are collected from each sample, and cell viability and sizes are determined. The detection threshold of the cell counter used here has a lower limit of 5 &#181;m.</li>
	<li>Transfer data from each run to a spreadsheet for further analysis.</li>
	<li>Calibrate the device on a regular basis using a 1 ml sample of 6 &#181;m and 8 &#181;m diameter polystyrene beads. 
	NOTE: The actual bead sizes measured by the manufacturer for each batch should be used, not the nominal size. 6 and 8 &#181;m beads are convenient because these diameters are close to the expected diameters of resting and activated T cells (see Figure 1).</li>
</ol>
6. Data and Statistical Analysis
<ol>
	<li>Analyze the data using appropriate statistical and graphing software. 
	NOTE: The spreadsheet for each run contains the number of cells with each diameter (range: 5 &#181;m to 70 &#181;m). The cells above a 17 &#181;m diameter are removed from the analysis to exclude dust particles and small bubbles, which can be read as viable cells by the instrument.</li>
	<li>Perform hypothesis testing using Welch&#39;s t-test for data sets with unequal variance; results are considered statistically significant if p &#60; 0.001. The formula used was, 
	<img alt="Equation 1" src="/files/ftp_upload/55212/55212eq1.jpg" /> 
	 
	where <img alt="Equation 2" src="/files/ftp_upload/55212/55212eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/55212/55212eq3.jpg" /> are sample means, <img alt="Equation 4" src="/files/ftp_upload/55212/55212eq4.jpg" /> and <img alt="Equation 5" src="/files/ftp_upload/55212/55212eq5.jpg" /> are sample variances, and <img alt="Equation 6" src="/files/ftp_upload/55212/55212eq6.jpg" /> and <img alt="Equation 7" src="/files/ftp_upload/55212/55212eq7.jpg" /> are sample sizes for each data set. The number of degrees of freedom is conservatively estimated using the smaller of the two sample sizes for each comparison. Bar graphs are presented as the mean &#177; SEM.</li>
</ol>
7. Splenic T-lymphocyte Proliferation Assay
<ol>
	<li>Measure T-cell proliferation using an MTS- or MTT-based colorimetric plate reader proliferation assay. 
	NOTE: The assay is based on MTS tetrazolium compound (Owen&#39;s reagent) reduction to soluble formazan product, likely by NADPH or NADH produced in metabolically active cells by dehydrogenase enzymes.</li>
	<li>Count purified T cells with a hemocytometer and re-suspend them in complete RPMI-1640 at a final density of 1 x 106 cell/ml. 
	NOTE: 1 mM DTT can be added to the cells at this stage.</li>
	<li>Activate the cells with PMA and ionomycin along with test drugs, if required, and mix gently (similar to step 4).</li>
	<li>Plate 100 &#181;l of the cells in each well of a 96-well cell culture treated plate and incubate at 37 &#176;C with 5% CO2 for 48 hr. Add 100 &#181;l of RPMI-1640 media without cells into one well to obtain a reading of the background absorbance.</li>
	<li>Add 20 &#181;l of MTS-based reagent to each well and incubate at 37 &#176;C with 5% CO2 for 4 hr.</li>
	<li>Take absorbance measurements at 490 nm using a plate reader. The absorbance measurements correspond to the number of metabolically active cells in each well. 
	NOTE: Subtract background levels if needed. The background absorbance values depend upon the type of culture medium, serum, external pH, and duration of MTS reagent exposure to light. MTS-based reagents are light-sensitive, and exposure to light for several hours may result in higher background absorbance values.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cells+by+mTOR:+the+known+knowns+and+the+known+unknowns.">Regulation of T cells by mTOR: the known knowns and the known unknowns.</a> Trends Immunol. 36 (1), 13-20 (2015).</li><li>Mandala, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+lymphocyte+trafficking+by+sphingosine-1-phosphate+receptor+agonists.">Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.</a> Science. 296 (5566), 346-349 (2002).</li><li>Qin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+and+FTY720+are+potent+inhibitors+of+the+transient+receptor+potential+melastatin+7+(TRPM7)+channels.">Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels.</a> Br J Pharmacol. 168 (6), 1294-1312 (2013).</li><li>Wulff, H., Kolski-Andreaco, A., Sankaranarayanan, A., Sabatier, J. M., Shakkottai, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulators+of+small-+and+intermediate-conductance+calcium-activated+potassium+channels+and+their+therapeutic+indications.">Modulators of small- and intermediate-conductance calcium-activated potassium channels and their therapeutic indications.</a> Curr Med Chem. 14 (13), 1437-1457 (2007).</li><li>Petho, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-proliferative+effect+of+cation+channel+blockers+in+T+lymphocytes+depends+on+the+strength+of+mitogenic+stimulation.">The anti-proliferative effect of cation channel blockers in T lymphocytes depends on the strength of mitogenic stimulation.</a> Immunol Lett. 171, 60-69 (2016).</li><li>Wulff, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+potent+and+selective+inhibitor+of+the+intermediate-conductance+Ca2+-activated+K++channel,+IKCa1:+a+potential+immunosuppressant.">Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.</a> Proc Natl Acad Sci USA. 97 (14), 8151-8156 (2000).</li><li>Lang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+cell+volume+regulatory+mechanisms.">Functional significance of cell volume regulatory mechanisms.</a> Physiol Rev. 78 (1), 247-306 (1998).</li><li>Hue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+liver+carbohydrate+and+fatty+acid+metabolism+by+cell+volume.">Control of liver carbohydrate and fatty acid metabolism by cell volume.</a> Biochem Soc Trans. 22 (2), 505-508 (1994).</li><li>O'Flaherty, J. T., Kreutzer, D. L., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+aggregation+and+swelling+induced+by+chemotactic+agents.">Neutrophil aggregation and swelling induced by chemotactic agents.</a> J Immunol. 119 (1), 232-239 (1977).</li><li>Hsu, L. S., Becker, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+changes+induced+in+rabbit+polymorphonuclear+leukocytes+by+chemotactic+factor+and+cytochalasin+B.">Volume changes induced in rabbit polymorphonuclear leukocytes by chemotactic factor and cytochalasin B.</a> Am J Pathol. 81 (1), 1-14 (1975).</li><li>Rosengren, S., Henson, P. M., Worthen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration-associated+volume+changes+in+neutrophils+facilitate+the+migratory+process+in+vitro.">Migration-associated volume changes in neutrophils facilitate the migratory process in vitro.</a> Am J Physiol. 267, C1623-C1632 (1994).</li></ol>

The authors have no competing financial interests.

Here, we describe a technique for the rapid detection and quantification of T-cell blastogenic transformation using an automated cell counter. Under our conditions (250 ng/ml PMA and 250 nM ionomycin stimulation), the cell surface area was increased two-fold and the volume three-fold after 48 hr of activation. The assay is sufficiently sensitive to detect blasting during the first 12 hr of activation, where the cell volume increased only 1.25-fold compared to resting (Figure 2D). A more physiologically relevant mechanism of T-cell activation using anti-CD3 and anti-CD28 antibody-coated magnetic beads produced a significant blastogenic response, with an average 2.3-fold increase in volume (72 hr after activation, Figure 3D). Human mononuclear cells showed a 2.6-fold change in volume upon PMA/ionomycin stimulation for 48 hr (Figure 4). The ICR mouse splenic T cell average diameter and volume were determined to be 6.9 &#181;m and 1.9 x 10-4 nl, respectively. Human PBMCs (from one healthy donor) had an average diameter of 7.7 &#181;m and an average volume of 2.7 x 10-4 nl. Using this protocol, we also tested increasing concentrations of PMA for their ability to activate T cells. These single-cell results were consistent with proliferation assays performed at similar PMA concentrations.
Several compounds reported to affect cell proliferation were tested: FTY72031,32; the immunosuppressants cyclosporine A, FK506, and rapamycin27,28; and TRAM-34, a blocker of calcium-activated KCa3.1 channels33,35. We found that, at the tested concentrations, the most potent inhibitors of blastogenesis were CsA and FK506. Rapamycin had a smaller but statistically significant suppressive effect on blastogenesis. TRAM-34, on the other hand, did not affect blastogenesis.
CsA suppressed both blastogenesis and proliferation, but not completely (Figure 2B and 2C), demonstrating that automated cell counter measurement is a good correlate of drug efficiency in T-cell proliferation. In the presence of rapamycin together with CsA, blastogenesis was completely inhibited, suggesting that NFAT- and mTOR-mediated pathways in combination can fully account for T-cell enlargement upon mitogenic stimulation with PMA/ionomycin (Figure 3C).
The dynamic range of some proliferation assays is limited (see Figure 2). Proliferation assays, such as the one we have used (Figure 2C), report a signal that includes contributions from apoptotic and necrotic cells. Automated diameter change measurements do not have that limitation, as the measurements pertain to viable cells only. Cell viability is assessed by the exclusion of trypan blue stain from healthy, viable cells. In size measurements, using forward light scatter in flow cytometry doublet cell discrimination can be problematic22. In the automated cell counter measurements, no doublet population was found (Figure 1). Also, the measurement was sufficiently precise to distinguish between the effects of 100 and 200 nM cyclosporine (Table 1) and to detect blastogenesis within 12 hr of mitogenic stimulation (Figure 2D).
This new assay is particularly useful for small numbers of samples. Up to 15 samples can be measured in 1 hr. However, the assay has its limitations, most of which can be mitigated. Even though it can effectively resolve small (&#60;1 &#181;m) difference in cell diameter, the machine model we used has a lower detection threshold of 5 &#181;m. This limit can cause the overestimation of very small cell sizes, since it effectively excludes all cells with smaller diameters. However, newer models of the cell counter have detection thresholds of ~2 &#181;m, which should alleviate this problem. If there is too much debris in the sample, the instrument treats them as viable cells. Additionally, air bubbles can occasionally get into the flow cell and cause distortion of the cell images captured by the machine. The distortion does not appear to affect the viability determination, but it can affect the measured diameters. Therefore, it is recommended that all images be checked by the experimenter for air bubbles before the data from each trial are accepted for analysis. Since the software only draws circles around the cells, diameters of non-spherical cells may be skewed towards the long axis, making the assay less suitable for non-spherical cells.
This assay is quick, taking approximately 4 min per sample, compared to proliferation assays, which require a few hours. Data collection is also rather simple using the software included with the device. The software can export measurement data to a spreadsheet for analysis. Finally, it is a single-cell, as opposed to a population, measurement; both blastogenesis and proliferation are measured; and it distinguishes between viable and dead cells simultaneously. It can be used to evaluate various mouse strains for their ability to mount an immune response. The assay can also be used successfully for the measurement of blastogenesis in T cells from human donors (Figure 4), and it can also be used in other cell types.
Cell volume increases are known to contribute to the regulation of metabolism: cell swelling stimulates glutamine-induced glycogen synthesis and lipogenesis in hepatocytes36,37. Neutrophils display migration-associated volume increases of 35-60%, while chemotactic agents induce 10-15% swelling38-40. The current assay can potentially be used to study these processes.

T lymphocytes are the primary cells responsible for adaptive immunity in mammals. It is known that they respond to specific antigenic peptides presented by MHC molecules on the surface of antigen-presenting cells. Upon activation of a cognate T-cell receptor (TCR), the cell enlarges in a process termed blastogenic transformation, or blastogenesis. This process is detectable in the first ~6 hr after the stimulus is applied1. During blastogenesis, the volumes of individual T cells increase 2- to 4-fold2-6. Lymphocytes begin to proliferate in a process called clonal expansion, the purpose of which is to generate as many clones of the antigen-specific TCR-bearing cells as possible. The progeny cells then exert their immunologic function by differentiating into cytotoxic (CD8+) or helper (CD4+) effector T lymphocytes. Thus, na&#239;ve T lymphocytes in human or mouse blood are in the G0 (resting) phase of the cell cycle and support minimal metabolic activity. Upon exposure to antigens or mitogens, T cells reenter the cell cycle, with a concomitant stimulation of transcription and protein synthesis7-10. Mitogens, such as phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulate the lymphocytes through the activation of protein kinase C (PKC) and Ca2+-dependent signaling pathways1. The activation of T cells with PMA/ionomycin bypasses the TCR signaling steps.
In vitro proliferation assays are widely used for the purpose of evaluating lymphocyte function and response to stimuli. Proliferation readings are typically taken one to three days after the start of T-cell stimulation and reflect the collective state of hundreds or thousands of cells. The potency of various mitogens and immunomodulatory drugs in vitro can be evaluated by simply measuring proliferation rates in the presence of these compounds. Some of these assays and their limitations are discussed below.
For direct cell number counting, the procedure is time consuming, with a high probability of operator errors.
For DNA synthesis, the 3H-thymidine incorporation assay measures DNA synthesis, but its major limitation is its radiotoxicity. A non-radioactive alternative is BrdU, but the range of linear response for the cell growth is limited, and antibody treatment is required, which increases the number of steps in the procedure11,12.
For metabolic activity, tetrazolium salts (MTT, MTS, XTT, and WST-1) and resazurin dye-based colorimetric assays report the general metabolic state of dividing cell populations. However, MTT is not soluble in the culture medium, requiring additional wash steps, thus incorporating errors in the measurement; XTT needs additional components to reduce efficiently; MTS-, WST-1-, and resazurin-based measurements are affected by the culture medium pH and its components serum, albumin or phenol red13-16. These assays do not measure the actual number of viable cells but rather estimate the combined enzyme activities. Therefore, the proliferation rate may not be accurately determined by metabolic assays because of the non-linear correlation between cell number and dye reduction12,17.
For measuring ATP concentration, T-cell activation-induced increases in ATP correlate with proliferation. However, elevation of intracellular ATP is one of the initial steps of T cell activation; many steps behind is the actual proliferation17,18.
For dye dilution assay, CFSE fluorescent dye stains cells by covalently binding to intracellular proteins. The dye shows a proliferation-dependent decrease in fluorescent intensity, which can track the number of cell divisions. However, because of covalent protein labeling, the functions of these proteins can be compromised. The dye is toxic to the cells at higher concentrations. At lower dye concentrations, however, the initial fluorescence intensity is reduced, decreasing the number of cell divisions that can be tracked. Additionally, after labeling with CFSE, there is a proliferation-independent ~50% loss of initial fluorescence during the first 24 to 48 hr period, which limits the dynamic range of this assay19,20.
Most of these assays reflect the collective state of large numbers of cells and require the treatment of the cells with fluorescent dyes. Necrotic and apoptotic cells might also contribute to these measurements, unless they are removed from the analysis by staining with chemicals or antibodies.
Lymphocyte blastogenesis can be evaluated by a variety of methods, such as optical microscopy or flow cytometry4,21,22. Here, we describe a rapid method for the measurement of T-cell sizes using an automated cell counter, which collects real-time cell images that are stored and can be re-analyzed at a later time. In addition to size measurements, this device provides precise cell numbers and the percentage of viable cells, as determined by trypan blue stain exclusion. The device used in this protocol is commercially available, and the manufacturer tested the precision of the instrument using three different instruments and several concentration and viability controls. Results of these studies demonstrated a coefficient of variance that was generally below 6%. As noted in the protocol, the device is calibrated on a regular basis with 6 &#181;m and 8 &#181;m diameter polystyrene beads. The advantages of using a cell counter to differentiate between resting T cells and T lymphoblasts based on cell diameter is the ease of use and the automated nature of the analysis. The software is capable of drawing a circle around each cell and calculating the cell diameter. Additionally, the images are visible to the operator, who can verify the accuracy of the instrument in identifying the cells and correctly drawing a circle around them. In terms of limitations, the instrument cannot per se differentiate between debris and cells; therefore, it is important that the operator views every image as it is being processed. There is a potential for incorporating air bubbles, which will decrease the number of usable fields for analysis; however, this is rare if the regular flushing maintenance is performed.
In this study, groups of splenic T lymphocytes were stimulated with ionomycin and increasing concentrations of PMA for 12-48 hr. PMA concentrations as low as 2 ng/ml induced both a robust blastogenic response and significant proliferation. Measurements of the effects of several drugs, such as the immunosuppressants cyclosporine A (CsA), FK506 (tacrolimus), and rapamycin (sirolimus), as well as ion channel blockers TRAM-34 and FTY720 (fingolimod), on blastogenesis demonstrated good agreement with reported effects on proliferation. The blastogenic response of human PBMCs to PMA/ionomycin and murine T-cell stimulation with anti-CD3 and anti-CD28 antibody-coated magnetic beads were also measured.
The cell counter assay quantifies both blastogenesis and the proliferation rate (cell density) simultaneously but separately, unlike the abovementioned methods, which look at a combination of these effects. The presented protocol provides a rapid and robust technique for evaluating the potency of mitogenic and immunomodulatory agents.

<table><tbody><tr><td>RPMI-1640&amp;nbsp;</td><td>Lonza</td><td>BW12-702F</td><td></td></tr><tr><td>40 &amp;mu;m nylon cell strainers&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>nylon wool fiber columns</td><td>Polysciences, Inc.&amp;nbsp;</td><td>21759-1</td><td></td></tr><tr><td>50 ml conical tubes&amp;nbsp;</td><td>The Lab Depot</td><td>TLD431697</td><td></td></tr><tr><td>6-well cell culture treated plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>96-well cell culture treated plates</td><td>Thermo Fisher Scientific</td><td>130188</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher BioReagents</td><td>BP231-100</td><td></td></tr><tr><td>CellTiter 96 AQueous One Solution Cell Proliferation Assay&amp;nbsp;</td><td>Promega</td><td>G3582</td><td>MTS based assay</td></tr><tr><td>Cyclosporine A</td><td>Sigma-Aldrich</td><td>30024</td><td></td></tr><tr><td>FK506</td><td>Cayman Chemical Company</td><td>104987-11-3</td><td></td></tr><tr><td>Rapamycin</td><td>Santa Cruz Biotechnology</td><td>sc-3504</td><td></td></tr><tr><td>TRAM-34</td><td>Sigma-Aldrich</td><td>T6700</td><td></td></tr><tr><td>FTY720</td><td>Sigma-Aldrich</td><td>SML0700</td><td></td></tr><tr><td>Dynabeads Mouse T-Activator CD3/CD28</td><td>Gibco</td><td>11456D</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)&amp;nbsp;</td><td>Acros Organics&amp;nbsp;</td><td>356150010</td><td></td></tr><tr><td>Ionomycin calcium salt</td><td>Sigma-Aldrich</td><td>I0634</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN1670049</td><td>100x stock</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>HyClone</td><td>SH30378.02</td><td>10x stock</td></tr><tr><td>1,4-dithiothreitol (DTT)&amp;nbsp;</td><td>Research Products International</td><td>12/3/3483</td><td>reducing agent</td></tr><tr><td>8 &amp;micro;m micro particle size standard&amp;nbsp;</td><td>Sigma-Aldrich</td><td>84192-5ML-F</td><td>Actual&amp;nbsp; 8.02 &amp;micro;m</td></tr><tr><td>6 &amp;micro;m micro particle size standard&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89756-5ML-F</td><td>Actual 6.084 &amp;micro;m</td></tr><tr><td>Single magnetic separation stand for 1.5 - 2 ml tube</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP772F5</td><td></td></tr><tr><td>Cell culture incubator</td><td>Forma Scientific&amp;nbsp;</td><td>3110</td><td></td></tr><tr><td>Synergy H1 hybrid reader&amp;nbsp;</td><td>Bio Tek</td><td>BTH1M</td><td></td></tr><tr><td>Vi-CELL cell viability analyzer&amp;nbsp;</td><td>Beckman Coulter</td><td>731050</td><td></td></tr></tbody></table>

rapid quantification of mitogen-induced blastogenesis in t lymphocytes for identifying immunomodulatory drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

We used this assay to compare lymphocyte blast formation during stimulation with PMA, a phorbol ester, and ionomycin, a calcium ionophore. Figure 1A shows the frequency distribution of diameters of resting and pharmacologically activated splenic T cells. Treatment of cells with PMA/ionomycin for ~2 days resulted in a significant shift in the median of the distribution towards bigger diameters (see, for example reference4). The number of cells with smaller diameters was accordingly reduced. In order to ascertain that our device reported the correct diameters, we performed two calibrations with polystyrene beads of 6 &#181;m and 8 &#181;m diameters (see Materials Table). Figures 1B and 1C show readings from activated T cells superimposed with an 8 &#181;m standard and resting T cells superimposed with a 6 &#181;m standard. Figure 1D shows the control bead sizes reported by the manufacturer plotted against the median diameters measured with our automated cell counter. It appeared that the 6 &#181;m size was slightly overestimated in our measurements, probably due to the measurement threshold of the instrument having a lower limit of 5 &#181;m. However, the standard deviation of the bead diameters calculated from the cell counter measurements was in agreement with the standard deviation reported by the manufacturer. We conclude from these experiments that this device can be used reliably to compare resting and mitogen-stimulated mouse T-cell sizes and that the device can accurately measure the actual cell diameters.
We then proceeded to test the dependence of the mean diameter increase on PMA concentration and found that, at a fixed ionomycin concentration of 250 nM, the PMA effect was not significantly changed by elevating its concentration from 2 to 250 ng/ml (Figure 2A). Using the MTS-based assay, we measured T-cell proliferation at 50 and 250 ng/ml PMA and found no appreciable difference (Figure 2C), in agreement with our cell diameter data. The calcineurin inhibitor drugs cyclosporine A (300 nM) and FK506 (1 nM) suppressed both blastogenesis (Figure 2B) and proliferation (Figure 2C). Inhibition was not complete in both cases, however. Measurements performed 12 hr after PMA/ionomycin addition showed a 7.2% and 6.8% increase in diameter for 50 ng/ml and 250 ng/ml PMA, respectively (Figure 2D). Size increases at these two concentrations were not significantly different, as was the case for 48 hr stimulation (compare to Figure 2A).
The cell diameter data collected from resting and activated T cells after 48 hr of PMA/ionomycin stimulation are summarized in Table 1. The mean surface area and volume of resting ICR mouse splenic T cells were 151.4 &#181;m2 and 1.9 x 10-4 nl, respectively. The mean surface area and volume of activated T cells were 300.6 &#181;m2 and 5.9 x 10-4 nl, respectively. The overall average diameter increase for all activated cells was 40.92% (Table 1).
We also tested other chemical compounds reported to be immunosuppressive: CsA, FK506, rapamycin, FTY720, and TRAM-34. In Figure 3, cell size measurements obtained in resting and activated T cells in the absence and presence of these drugs are shown. CsA and FK506, which target the calcineurin-dependent nuclear factor of activated T cells (NFAT)27,28, were the most potent, inhibiting the blastogenic response by nearly 72% (Figure 3A and 3B). Interestingly, with increased PMA concentrations, the potency of CsA and FK506 dropped slightly, even though there was no additional cell diameter increase in the absence of these drugs (Figure 2A and 2B). Rapamycin, an immunosuppressant that inhibits mammalian target of rapamycin (mTOR)29,30, had a moderate but statistically significant effect (Figure 3A, 3B and 3C). FTY720 is a sphingosine 1-phosphate receptor agonist that inhibits lymphocyte egress into circulation31 and was recently found to inhibit TRPM7, a cation channel highly expressed in T lymphocytes32. Both FTY720 and rapamycin had a comparable effect on blastogenesis, reducing it from the average of a 52% increase in diameter to approximately 34% (Figure 3A and 3B). TRAM-34 is a blocker of the calcium-activated potassium channel KCa3.133. Tested at 700 nM, TRAM-34 was ineffective in murine T cells, in accordance with a recent human T-cell proliferation study; its potency may depend on the nature and strength of mitogenic stimulation34 (Figure 3A and 3B). 700 nM TRAM-34 was effective in blocking KCa3.1 channels in our patch clamp electrophysiology experiments (data not shown). This comparison was made between resting and drug-exposed cells in multiple trials within the same experiment at 48 hr. When rapamycin and CsA were used together, blastogenesis was completely inhibited (Figure 3C).
Activation of murine T cells with anti-CD3/anti-CD28-coated magnetic beads for 72 hr produced a 27% increase in the average diameter, which was reduced to 5% in the presence of CsA (Figure 3D). Human mononuclear cells upon PMA/ionomycin activation for 48 hr increased in diameter by 33%, and activation in the presence of CsA decreased the response to 23% (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/55212/55212fig1.jpg" /> 
Figure 1: Frequency distributions of murine splenic T-cell diameters. (A) Black columns indicate resting and red columns indicate PMA/ionomycin-activated (48 hr) cells. (B) Distributions of activated splenic T cells superimposed on an 8 &#181;m particle size calibration standard. (C) Distributions of resting T cells superimposed on a 6 &#181;m calibration standard. The numbers of cells and beads used are indicated in boxes. (D) Median diameter measured by the cell counter plotted against the bead diameter reported by the manufacturer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55212/55212fig2.jpg" /> 
Figure 2: Dependence of murine T-cell activation on PMA concentration. (A) Mean diameters of T cells either untreated or treated with 2, 50, and 250 ng/ml PMA at a fixed ionomycin concentration (250 nM). (B) Cell counter measurements of resting and activated (2, 50, 250 ng/ml PMA) T cells in the absence and presence of 300 nM CsA or 1 nM FK506. (C) MTS proliferation assay at 50 and 250 ng/ml PMA with 250 nM ionomycin in the absence and presence of 300 nM CsA. Significant differences (p &#60; 0.001) are indicated with asterisks. n is the total number of trials. Data are from cells isolated from 3 mice. (D) Cell diameters of resting and activated T cells (250 ng/ml PMA and 250 nM ionomycin) 12 hr after activation in the absence and presence of 300 nM CsA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55212/55212fig3.jpg" /> 
Figure 3: Effects of CsA, FK506, FTY720, rapamycin, and TRAM-34 on cell size. (A and C) Average diameters of resting and PMA/ionomycin-activated murine T cells in the absence and presence of drugs. Concentrations are shown in the box. (B) Data from A expressed as the percent increase over the resting T-cell diameter. Lymphocyte activation was performed with 250 ng/ml PMA and 250 nM ionomycin, and the measurements were taken after 48 hr. (D) Cell counter measurements of resting and anti-CD3/CD28-activated T cells, 72 hr after activation in the absence and presence of 300 nM CsA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55212/55212fig4.jpg" /> 
Figure 4: Blastogenesis in human PBMCs upon mitogen stimulation. (A) Black columns indicate resting and red columns indicate activated PBMCs. (B) Mean diameters of resting and activated PBMCs in the absence and presence of 300 nM CsA. Cells were activated with 250 ng/ml PMA and 250 nM ionomycin, and the measurements were taken after 48 hr of activation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55212/55212fig4large.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>Column1</td>
			<td>Resting</td>
			<td>Activated</td>
			<td>CsA 100 nM</td>
			<td>CsA 200 nM</td>
		</tr>
		<tr>
			<td>Average Diameter</td>
			<td>6.94 &#181;m</td>
			<td>9.78 &#181;m</td>
			<td>8.19 &#181;m</td>
			<td>7.92 &#181;m</td>
		</tr>
		<tr>
			<td>Average Increase in Diameter</td>
			<td></td>
			<td>40.92%</td>
			<td>17.88%</td>
			<td>14.09%</td>
		</tr>
		<tr>
			<td>Average Surface Area</td>
			<td>151.37 &#181;m&#178;</td>
			<td>300.61 &#181;m&#178;</td>
			<td>210.56 &#181;m&#178;</td>
			<td>197.22 &#181;m&#178;</td>
		</tr>
		<tr>
			<td>Average Increase in Surface Area</td>
			<td></td>
			<td>98.59%</td>
			<td>39.00%</td>
			<td>30.16%</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of the average cell diameter and relative increases in surface area. Measurements were performed 48 hr after activation with 250 ng/ml PMA and 250 nM ionomycin.

Watch this Scientific Journal Video about Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs at JoVE.com

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

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Immunology and Infection

18.8K Views.  Wright State University. T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters. 

Video: Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Cancer Research

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

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.

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.

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

Medicine

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. ...

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

Source: Fouda, A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55199">A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.</a> J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds&#39; stimulatory and inhibitory effects, respectively.

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

	Perform all steps under a 70% ethanol-cleaned biological hood.
	Remove 70 mL of pre-warmed ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

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 Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

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